Insect Molecular Biology
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Insect Molecular Biology (2014) 23(3), 367–380
doi: 10.1111/imb.12087
Telomeric transcriptome from Chironomus riparius (Diptera), a species with noncanonical telomeres
J. L. Martínez-Guitarte*‡, M. de la Fuente†‡ and G. Morcillo* *Grupo de Biología y Toxicología Ambiental. Facultad de Ciencias. Universidad Nacional de Educación a Distancia, UNED. Madrid. Spain; †Departamento de Ciencias y Técnicas Fisicoquímicas. Facultad de Ciencias. Universidad Nacional de Educación a Distancia, UNED. Madrid. Spain
scripts between control and stressful environmental conditions, supporting the idea that telomeric RNAs could have a relevant role in cellular metabolism in insect cells. Keywords: insect telomeres, non-telomerase telomere, non-coding RNA, heat shock, transcription regulation.
Abstract
Introduction
Although there are alternative telomere structures, most telomeres contain DNA arrays of short repeats (6–26 bp) maintained by telomerase. Like other diptera, Chironomus riparius has noncanonical telomeres and three subfamilies, TsA, TsB and TsC, of longer sequences (176 bp) are found at their chromosomal ends. Reverse transcription PCR was used to show that different RNAs are transcribed from these sequences. Only one strand from TsA sequences seems to render a noncoding RNA (named CriTER-A); transcripts from both TsB strands were found (CriTER-B and αCriTER-B) but no TsC transcripts were detected. Interestingly, these sequences showed a differential transcriptional response upon heat shock, and they were also differentially affected by inhibitors of RNA polymerase II and RNA polymerase III. A computer search for transcription factor binding sites revealed putative regulatory cis-elements within the transcribed sequence, reinforcing the experimental evidence which suggests that the telomeric repeat might function as a promoter. This work describes the telomeric transcriptome of an insect with nontelomerase telomeres, confirming the evolutionary conservation of telomere transcription. Our data reveal differences in the regulation of telomeric tran-
Telomeres are specialized nucleoprotein structures with repetitive DNA sequences that cap the ends of linear chromosomes. They are essential for completion of linear DNA replication, as well as for maintaining chromosome stability, and have a crucial role in regulating cellular senescence. Recent studies have also causally linked telomeres with many genetic, metabolic and inflammatory human diseases (Ding et al., 2013; Kong et al., 2013). Most eukaryotic telomeres are formed by simple and short repeats (6–26 bp) maintained by the enzyme telomerase, a reverse transcriptase. Nevertheless, a number of exceptions, with different types of telomeric DNA at their chromosome termini, have been described mainly in arthropoda and plants (Zellinger & Riha, 2007; Capkova Frydrychova et al., 2008). There are alternative telomere structures and alternative mechanisms to telomerase for telomere maintenance in insects (Biessmann & Mason, 2003). Among these, Chironomus (Diptera: Chironomidae) telomeres represent an interesting model as neither the short telomerase repeats (up to 30bp) nor the long LINE-like retrotransposons (HeT-A, TART, TAHRE, up to 4Kb), which are typical of Drosophila (Diptera: Drosophilidae) telomeres (Abad et al., 2004; Casacuberta & Pardue, 2005; Mason et al., 2008) are found. Instead, long arrays of DNA repeats, which are size (170–350 bp) and sequence species-specific, constitute the chromosome termini (Carmona et al., 1985; López et al., 1996, 1996; Rosén & Edström, 2000). These complex repetitive sequences must be functionally homologous to the other types of telomeric DNA and involved in the telomere-maintenance mechanism. The
First published online 18 February 2014. Corresponding author: José-Luis Martínez-Guitarte. Facultad de Ciencias. UNED. Senda del Rey 9. 28040 Madrid. Spain. Tel.: +34 91 398 7644; fax: +34 91 397 6697; e-mail: [email protected] ‡These authors contributed equally to this paper.
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localization of reverse transcriptase in Chironomid telomeres (López et al., 1999; Gorab, 2003; Díez et al., 2006) suggests that retrotranscription processes are also involved in the maintenance mechanism of these atypical telomeres. Curiously, composite structures of a retrotransposon and a complex repeat associated with chromosome ends have been found in Rhynchosciara [Diptera: Sciaridae (Madalena & Gorab, 2005; Madalena et al., 2010; Fernandes et al., 2012)]. Telomeres had been traditionally considered as transcriptionally inactive regions of chromosomes because of the lack of protein-coding genes in these regions, the heterochromatic state of the chromatin and the silencing of genes inserted into telomeric domains (telomere position effect). The discovery of telomeric repeat-containing RNAs (named TERRA) has challenged the view of transcriptionally silent telomeres (Azzalin et al., 2007; Schoeftner & Blasco, 2008) and redefined the telomere as a complex structure consisting not only of DNA and proteins but also RNA. As a result, research on telomere transcription has increased dramatically in recent years and the function of telomere transcripts is now the subject of intensive study, because of its possible role in the function of the telomere itself and the potential connections with aging and cancer (Caslini, 2010; Bah & Azzalin, 2012). TERRA RNA molecules have been reported in a number of eukaryotes with telomerase repeats, including mammals, birds, zebrafish, budding yeast, fission yeast and Arabidopsis (Solovei et al., 1994; Luke et al., 2008; Vrbsky et al., 2010; Bah & Azzalin, 2012; Bah et al., 2012; Greenwood & Cooper, 2012). The set of transcripts derived from telomeric and subtelomeric sequences has been defined as the telomeric transcriptome (Bah & Azzalin, 2012). In telomeres, transcription from both strands has been demonstrated only in fission yeast, where besides TERRA an anti-sense transcript (named ARIA) is produced together with sense and antisense subtelomeric transcripts (Bah et al., 2012; Greenwood & Cooper, 2012). The functions associated with the telomeric transcriptome still remains elusive. In humans and rodents a fraction of TERRA associates with telomeric heterochromatin and it has been proposed that it is involved in cancer progression, telomerase regulation, replicative senescence and epigenetic protection of telomeric DNA (Caslini, 2010). Telomeric transcriptional activity has also been detected in non-telomerase-dependent insect species. Drosophila telomeres, which are formed by non-long-terminal-repeat transposons, are in fact retrotranscribed to maintain the telomere length (Mason et al., 2008). Chironomus riparius, also known as Chironomus thummi, is the only species in which environmentally regulated telomeric transcription has been reported. Decades ago, it was established that its telomeres transcribe upon heat shock
(Morcillo et al., 1981). Interestingly, signals of telomere transcription, such as the existence of 350 nm ribonucleoprotein ethylenediaminetetraacetic acid (EDTA)positive particles and local labelling after in vivo [3H] uridine pulses, were clearly detected when telomeres were activated forming a giant puff under heat shock and other stressful conditions (Santa-Cruz et al., 1984; Barettino et al., 1988). Recently, using a high urea RNA extraction method, we detected the telomeric transcript by reverse transcription (RT)-PCR showing that it is a long noncoding RNA which is transcribed in a regular way and modulates its expression in response to different stimuli such as heat shock or specific chemicals, such as bisphenol A (Martínez-Guitarte et al., 2008, 2012). The telomeric RNA of C. riparius has been included in the group of noncoding RNAs as it does not show an open reading frame and in Northern blot it is detected as a ladder from 176 bp to >1000 bp (Martínez-Guitarte et al., 2008). The telomeres of this species show three variants of a 176 bp sequence, named TsA, TsB, and TsC, which differ in ∼10% of the sequence (Martinez et al., 2001a). We were able to show that TsA and TsB have a binding site for the heat shock factor, providing a molecular basis for telomere transcription upon heat shock (Martinez et al., 2001b), but the very nature of the transcripts remains unknown. In the present work, the telomeric transcriptome of C. riparius was analysed in an attempt to characterize the transcriptional activity in non-telomerase telomeres. Using RT-PCR with specific primers for the different telomeric sequences, the transcription profile was elucidated, finding three different transcripts of the telomere sequences. In addition, transcriptional behaviour during heat shock and the effects of different transcription inhibitors were analysed. The results support the idea that transcriptional activity in telomeres is under complex regulation and is altered under stressful environmental conditions. Bioinformatic analysis of telomeric sequences revealed putative transcription regulation sites within the telomeric repeats themselves, providing support for their different transcriptional behaviour. This work provides new insights into the telomeric transcriptome of a nontelomerase insect species and its complex regulation, thereby adding to the growing body of evidence regarding the existence of noncoding telomeric RNAs that might have a relevant role in cell metabolism. Results Analysis of Chironomus riparius telomeric RNA under control and heat shock conditions To analyse the variety of RNAs produced from the telomeres, specific primers were designed to retrotranscribe and detect by PCR each one of the strands of the three known telomeric sequences (TsA, TsB and TsC) © 2014 The Royal Entomological Society, 23, 367–380
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Table 1. Primers used in retrotranscription and in PCR. rpL13 (EF179386), TsA (M33211.1), TsB (AJ295633), and TsC (AJ295634) sequences from GenBank database were used to design the primers. The size is that obtained by reverse transcription-PCR in each case with the primers indicated Primer
Sequence
Method
Tel Retro M13 F Tel Retro B M13F L13 R L13 F L13 R2 M13 F RTPCR TsA R RTPCR TsB R RTPCR TsC R Tel RTPCR M13F B RTPCR TsA R B RTPCR TsB R B RTPCR TsC R B
5′-GTAAAACGACGGCCAGTAGATGCATCCAACG-3′ 5′-TGTTTTCCCAGTCACGACCGTTGGATGCATCT-3′ 5′-TTGGCATAATTGGTCCAG-3′ 5′-AACGTGCTTTCCCAAGAC-3′ 5′-TCATGAACTGGGAAGAGG-3′ 5′-GTAAAACGACGGCCAGT-3′ 5′-AAAATTTTCATGTTTTCG-3′ 5′-CACAGAATTGAAGTTTTTC-3′ 5′-AAAAGATTTCAACCTCTTC-3′ 5′-TGTTTTCCCAGTCACGAC-3′ 5′-CGAAAACATGAAAATTTT-3′ 5′-GAAAAACTTCAATTCTGTG-3′ 5′-GAAGAGGTTGAAATCTTTT-3′
RT RT RT PCR PCR PCR PCR PCR PCR PCR PCR PCR PCR
from C. riparius (Table 1). When RT-PCR was carried out, RNAs with primers specific for TsA and TsB sequences were detected (Fig. 1A) from only one strand for TsA, and from both strands for TsB; however, no signal was obtained with any of the specific primers for TsC sequences (data not shown). We named the RNAs retrotranscribed with Tel Retro M13 F primer as CriTER-A (Chironomus riparius TElomeric RNA from TsA) and CriTER-B, respectively, while the one observed when retrotranscription was carried out with Tel Retro B M13F was called αCriTER-B. The α indicates that the complementary strand of TsB was used as a template when producing this RNA. These results show that the telomeric transcriptome of C. riparius is formed by at least three different RNAs, produced from TsA and TsB sequences. Under normal growing conditions for larvae, the level of CriTER-A is much lower when compared with the RNAs transcribed from TsB sequences, which are much more abundant. Under environmentally stressful conditions, such as heat shock, CriTER-A transcripts dramatically increase. Treatment of fourth instar larvae for 2 h at 35 °C showed that CriTER-A levels increased fourfold in comparison with control larvae, whereas CriTER-B levels were only slightly increased, as were αCriTER-B RNA levels (Fig. 1B). No transcripts complementary to any of the TsC strands were found after the temperature shock (data not shown). These results show the differential transcriptional behaviour of the different telomeric repeats that, despite a rather similar sequence (∼10% of differences), revealed one strand of TsA as the main source for the telomeric RNA increase upon heat shock. TsB showed transcription in both strands, but with a lower activity in heat shock conditions. Effect of different RNA polymerase inhibitors on telomeric transcripts As RNA polymerase II and RNA polymerase III have been associated with the transcription of several long stress© 2014 The Royal Entomological Society, 23, 367–380
Size
262 bp 90 bp
119 bp
Figure 1. Telomeric RNAs and their transcription in heat shock conditions. (A) Detection of transcripts from TsA and TsB with specific primers. Total RNA was isolated and used to perform the retrotranscription with oligonucleotides to detect each strand. PCR was carried out with specific primers, and the transcripts were detected by electrophoresis in acrylamide gel. TsX + and TsX – notate strands retrotranscribed with Tel Retro M13 F and with Tel Retro B M13F oligonucleotides respectively. (B) Detection of CriTER-A, CriTER-B and αCriTER-B in control (C) and heat-shocked (HS) fourth instar larvae. The ribosomal protein L13 (rpL13) was used as reference gene. Gel images and quantification are shown. Values were normalized against rpL13. Means and standard deviations are shown from three independent experiments.
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responsive noncoding RNAs, we used different inhibitors to elucidate whether or not telomeric transcription and the different telomeric transcripts identified could be affected by them, both in control and heat shock conditions. Fourth instar larvae were in vivo exposed to the following: actynomicin D (AMD), a known general transcription inhibitor; 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), a specific inhibitor for RNA polymerase II; and (N-[1(3-(5-Chloro-3-methylbenzo[b]thiophen-2-yl-1-methyl-1Hpyrazol-5-yl)]-2-chlorobenzenesulfonamide), a specific inhibitor for RNA polymerase III (Bensaude, 2011; Ljungman, 2012). Figure 2A shows that, under normal growing conditions, AMD does not alter CriTER-A levels, but it decreases the levels of both transcripts of TsB. When heat shock-treated larvae were analysed, it was seen that AMD caused a general decrease of the three RNA levels. Yet, in a control situation, DRB only produced a decrease in CriTER-B levels, similar to that observed with AMD, whereas in heat shock-treated larvae, both RNAs showed a decrease in their levels, but to a lesser extent than with AMD. Levels of αCriTER-B were practically unaltered by the inhibitor, both in control conditions and in heat shocked larvae. Finally, with the Pol III inhibitor (Fig. 2B), CriTER-A, CriTER-B and αCriTER-B did not show any decrease in control larvae. Nevertheless, when heat-shocked larvae were analysed, a different pattern was observed. Although CriTER-A levels were not affected, CriTER-B and αCriTER-B levels suffered a sharp decline, especially strong for αCriTER-B (∼40%, as shown in Fig. 2C). The inhibition experiments suggest that RNA polymerase II is involved in the transcription of TsA and both strands of the TsB sequence, whereas RNA polymerase III has a relevant role in the synthesis of CriTER-B and αCriTER-B under heat shock conditions.
Computational analysis of promoters of the telomere sequence Our research group previously demonstrated that there is a heat shock element (HSE) in TsA and TsB sequences (Martinez et al., 2001b). This supports the hypothesis that regulation of telomeric transcription could depend on an internal promoter. Moreover, RNA polymerase inhibitors have shown that RNA polymerase II and RNA polymerase III could be involved in transcription at telomeres. To detect potential transcription factor binding sites related with Pol II and/or Pol III-type promoters, MATINSPECTOR software was used to predict the potential transcription factor binding sites in both strands of telomeric sequences. It is well established that presence of individual binding sites are not sufficient to indicate transcriptional function; however, comparative analysis of the putative elements appearing in each sequence provided
interesting results, which may be related to their differential transcriptional behaviour. For this analysis, CriTER-A and CriTER-B were the sense transcripts so they were considered plus strand, whereas the complementary one was considered to be antisense and then minus strand. A graphical summary of the more relevant results is presented in Figs 3 and 4. When default parameters were used, matches for core promoter elements were only found in TsA sequences (Supporting Information). The presence of a core promoter initiator element with high matrix similarity (0.952, where a perfect match would be 1.000) must be highlighted (Fig. 5). The most commonly occurring sequence motif in core promoters is probably Inr, which is recognized by TFIID, a transcription factor for Pol II, and this binding seems to be particularly important in the initial steps of transcription. It includes the transcription start site and is present in both TATA-containing and TATA-less promoters. In Drosophila promoters, its consensus sequence is TCAKTY, with the underlined A often being the +1 start site position (Kutach & Kadonaga, 2000; Smale & Kadonaga, 2003). If a lower matrix similarity threshold is used, other good matches for Inr elements can be observed (matrix similarity >0.80, Fig. 5), appearing in both strands of TsB but only in the TsA-plus strand. Curiously, these are the same strands that have been detected for producing RNA. Only one other significant core promoter element worth highlighting appeared in this analysis, this was a putative TATA box appropriately positioned (29 bp upstream of the transcription start site) in minus TsB. This strand had a resemblance in its architecture to a characteristic Pol II promoter (Fig. 3). The MATINSPECTOR program also predicted other worthwhile regulatory sequences by matching with insect matrix library. First, a HSE was detected in TsA and TsB (with matrix similarity up to 0.892), but not in the TsC sequence. Our group had previously demonstrated that it binds heat shock factor (HSF), a regulatory element associated with heat shock (Martinez et al., 2001b). Moreover, a putative recognition element for insulator factors boundary element-associated factor (BEAF)/DNA replication related element-binding factor (DREF) was predicted in both strands of TsA (matrix similarity up to 0.988). A manual motif search for consensus sequences of other relevant elements revealed a putative binding site for GAGA factor only in TsA-plus and a TC-box in both strands of TsA. This TC-box can bind a TBP-related factor specific to insects, called TRF1, which interacts with TFIIA and TFIIB and is also a component of TFIIIB (Smale & Kadonaga, 2003). As mentioned above, the different telomeric sequences of C. riparius are rather similar, with ∼90% similarity between them; however, the differences are not homogeneously distributed and, as shown in Fig. 3, there is one region © 2014 The Royal Entomological Society, 23, 367–380
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Figure 2. Effects of RNA synthesis inhibitors in control and heat shock conditions. Inhibitors of transcription reduce the RNA levels of telomeric transcripts. (A) Actinomycin D (AMD) and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) treatments were used to analyse the inhibition of the RNA polymerase II in RNA levels of CriTER-A, CriTER-B and αCriTER-B in control (C) and heat-shocked (HS) fourth instar larvae. (B) Changes for CriTER-A, CriTER-B and αCriTER-B levels in control (C) and heat shocked (HS) fourth instar larvae RNA levels in the presence of a specific RNA polymerase III inhibitor (RPIII inh). Values were normalized against ribosomal protein L13 (rpL13). Gel images and quantification are shown. Means and standard deviations are shown from three independent experiments. (C) Summary table with the decrease of RNA levels observed in each case as a percentage relative to the reference situation (control or heat shock).
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Figure 3. RNA polymerase II-related motifs in the telomeric sequences of Chironomus riparius. RNA polymerase II motifs are shown in the telomeric sequences and in a graphical map of each one. Location in plus (A) and minus (B) strands is shown. Vertical lines in the top of the sequences are for each 10 nucleotides, while the dots indicate the identity between the sequences. The last 17 bases of TsA (M33211.1), TsB (AJ295633), and TsC (AJ295634) sequences from the GenBank database were moved to the front to maintain heat shock element (HSE) together. Minus strands were obtained using the Sequence Manipulation Suite (SMS2, http://www.bioinformatics.org). BEAF, boundary element-associated factor; DREF, DNA replication related element-binding factor.
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Figure 4. RNA polymerase III-related motifs in the telomeric sequences of Chironomus riparius. RNA polymerase III motifs are shown in the telomeric sequences and in a graphical map of each one. Location in plus (A) and minus (B) strands is shown. Vertical lines in the top of the sequences are for each ten nucleotides, while the dots indicate the identity between the sequences. The last 17 bases of TsA (M33211.1), TsB (AJ295633) and TsC (AJ295634) sequences from GenBank database were moved to the front.
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Figure 5. Putative Inr elements found by MatInspector in the telomeric sequences. The different putative Inr elements are shown in a graph with their relative position to putative important sites in transcription by RNA polymerase II. The position of the putative Transcription start site is indicated as a bold A. In the table, the matrix similarity shows the probability of the Inr according to MatInspector. The position of each Inr is indicated in reference to sequences presented in Fig. 3: (+): plus strand, (−): minus strand.
∼30 bp towards the end of the plus strand that accumulates 50% of the dissimilarities. Two Inr in TsB are located in this region, but are absent in TsA because the differences observed turn them into a putative binding site for GAGA factor. It has been suggested that the GAGA factor acts as an anti-repressor by helping to disrupt nucleosomes, and it has also been related to heat shock genes and paused Pol II promoters (Shopland et al., 1995). Analysis of the other region that accumulates several differences showed a similar situation, in which the variations between the sequences lead to another distinctive
element in TsA, the recognition element for BEAF/DREF (predicted using the MATINSPECTOR program, with matrix similarity up to 0.988). The insulator factor BEAF seems to bind to DNA and regulates the expression of genes involving nucleosome-positioning conformations at promoters, which are coupled with RNA polymerase II pausing (Mellor, 2010; Gonsalves et al., 2011). Although there is a great diversity of RNA pol III promoters, there are two featured elements, boxes A and B, which bind the TFIIIC factor (Orioli et al., 2012). A manual search was carried out, which showed that both strands of © 2014 The Royal Entomological Society, 23, 367–380
Telomeric transcriptome from Chironomus riparius TsB and TsC have putative sites (Fig. 4). Yet, TsA presented putative A-boxes in both strands, whereas only its plus strand showed B-boxes. Regarding the termination signal formed by four T residues, as telomeric sequences of C. riparius are rich in AT pairs, there are several of these termination signals in all the sequences. The performed theoretical analysis showed that there are putative regulation elements in both TsA and TsB (Fig. 5), suggesting a possible molecular basis that could explain their transcription. In addition, this analysis suggested that the TsB-minus structure resembles a typical RNA Pol II promoter. Variations in key motif could be related to a different architecture in TsA, in which these motifs would be associated with clearing the region of nucleosomes to promote transcription (Mellor, 2010). Although RNAs from TsC or the minus strand of TsA have not been detected, the presence of putative binding sites opens up the possibility that they could be transcribed in other non-tested conditions. Discussion The present study reports the telomeric transcriptome of an insect with non-telomerase maintained telomeres. Three different RNAs, CriTER-A, CriTER-B and αCriTER-B, were identified in C. riparius that are transcribed from the telomeric DNA repeats TsA and TsB. The cellular pattern of telomeric transcripts differed in environmentally stressful conditions from that in the control situation. The levels of CriTER-A increased up to fourfold during heat shock. It is worth noting that, in contrast to TsA and TsB repeats, the TsC sequences did not appear to be transcribed. In addition, it was found that transcription inhibitors of RNA pol II and RNA pol III affect the set of transcripts derived from telomeric repeats. A bioinformatic analysis revealed the existence of putative promoter regulatory sequences within these transcribed repeats. These findings provide new insights into the telomeric RNA expression of C. riparius and open a new perspective of the transcriptional activity of telomeres, widening the field to those telomeres bearing telomerase-independent repeats. Long noncoding RNAs at eukaryotic telomeres are likely to be more prevalent than previously thought. The variety of telomeric RNAs found in C. riparius is in accordance with previous data, particularly from humans and yeasts, where the telomeric transcriptome has been thoroughly characterized and different transcripts from telomere sequences have been identified. In mammalian cells and budding yeast, TERRA transcripts contain G-rich telomeric repeats and tracks of adjacent subtelomeric sequences, whereas C-rich transcripts complementary to TERRA have not been detected (Azzalin et al., 2007; Luke et al., 2008; Schoeftner & Blasco, 2008). In addition, budding yeast contains ARRET RNA molecules, which are © 2014 The Royal Entomological Society, 23, 367–380
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complementary to the subtelomeric tract of TERRA but devoid of telomeric repeats (Luke et al., 2008). TERRA and ARRET have also been detected in Arabidopsis (Vrbsky et al., 2010). Furthermore, fission yeast telomeres also produce ARIA, a C-rich telomeric repeat-containing RNA, and α-ARRET that are subtelomeric transcripts complementary to ARRET (Bah et al., 2012; Greenwood & Cooper, 2012). In insects, only Drosophila telomeric transcriptome has been analysed in detail. Sense transcripts from the telomeric elements, HeT-A, TART and TAHRE, include open reading frames for proteins involved in their retrotransposition (Capkova Frydrychova et al., 2008); however, it has been described that TART and HeT-A are bidirectionally transcribed (Danilevskaya et al., 1999; Maxwell et al., 2006; Shpiz et al., 2009), showing a similar situation to mammals and yeasts. As in fission yeast, in C. riparius transcripts containing only telomeric repeats (ARIA) and transcripts derived from both strands have been identified, although in this latter case (yeast ARRET and α-ARRET) are derived from subtelomeric sequences. Although the transcript size for CriTER-A, CriTER-B and αCriTER-B corresponds to the telomeric monomer (176 bp), dimers, trimers and a smear denoting heterogeneous sizes and up to 10 Kb signals have also been previously detected for telomeric RNA in Northern blot after a high urea RNA extraction method (Martínez-Guitarte et al., 2008). The population of TERRA molecules is also very heterogeneous in length, ranging from ∼100 up to 9000 bases in mammals while it is shorter in yeast (0.80, searches with a decreased matrix similarity threshold were also performed to find more matches in this range. The DiProGB tool (Friedel et al., 2009) and MEGA software (Tamura et al., 2011) for motif search were also used to search for consensus sequences of specific core elements.
Funding This work was supported by Ministerio de Economia y Competitividad, Spain [grant CTM-2012-37547 from the Ciencias y Tecnologías Medioambientales programme].
Acknowledgements The authors wish to thank Dr T. Carretero (University of Zaragoza) and Ted Cater for critical reading of the manuscript.
Conflict of interest None declared.
References Abad, J.P., De Pablos, B., Osoegawa, K., De Jong, P.J., Martín-Gallardo, A. and Villasante, A. (2004) TAHRE, a novel telomeric retrotransposon from Drosophila melanogaster, reveals the origin of Drosophila telomeres. Mol Biol Evol 21: 1620–1624. Azzalin, C.M., Reichenbach, P., Khoriauli, L., Giulotto, E. and Lingner, J. (2007) Telomeric repeat containing RNA and RNA surveillance factors at mammalian chromosome ends. Science 318: 798–801. Bah, A. and Azzalin, C.M. (2012) The telomeric transcriptome: from fission yeast to mammals. Int J Biochem Cell Biol 44: 1055–1059. Bah, A., Wischnewski, H., Shchepachev, V. and Azzalin, C.M. (2012) The telomeric transcriptome of Schizosaccharomyces pombe. Nucleic Acids Res 40: 2995–3005. Balk, B., Maicher, A., Dees, M., Klermund, J., Luke-Glasser, S., Bender, K. et al. (2013) Telomeric RNA-DNA hybrids affect
telomere-length dynamics and senescence. Nat Struct Mol Biol 20: 1199–2205. Barettino, D., Morcillo, G. and Díez, J.L. (1988) Induction of the heat-shock response by carbon dioxide in Chironomus thummi. Cell Differ 23: 27–36. Bensaude, O. (2011) Inhibiting eukaryotic transcription: which compound to choose? How to evaluate its activity? Transcription 2: 103–108. Biessmann, H. and Mason, J.M. (2003) Telomerase-independent mechanisms of telomere elongation. Cell Mol Life Sci 60: 2325–2333. Capkova Frydrychova, R., Biessmann, H. and Mason, J.M. (2008) Regulation of telomere length in Drosophila. Cytogenet Genome Res 122: 356–364. Carmona, M.J., Morcillo, G., Galler, R., Martinez-Salas, E., Campa, A.G., Diez, J.L. et al. (1985) Cloning and molecular characterization of a telomeric sequence from a temperatureinduced Balbiani ring. Chromosoma 92: 108–115. Cartharius, K., Frech, K., Grote, K., Klocke, B., Haltmeier, M., Klingenhoff, A. et al. (2005) MatInspector and beyond: promoter analysis based on transcription factor binding sites. Bioinformatics 21: 2933–2942. Casacuberta, E. and Pardue, M.-L. (2005) HeT-A and TART, two Drosophila retrotransposons with a bona fide role in chromosome structure for more than 60 million years. Cytogenet Genome Res 110: 152–159. Caslini, C. (2010) Transcriptional regulation of telomeric noncoding RNA: implications on telomere biology, replicative senescence and cancer. RNA Biol 7: 18–22. Danilevskaya, O.N., Arkhipova, I.R., Traverse, K.L. and Pardue, M.L. (1997) Promoting in tandem: the promoter for telomere transposon HeT-A and implications for the evolution of retroviral LTRs. Cell 88: 647–655. Danilevskaya, O.N., Traverse, K.L., Hogan, N.C., DeBaryshe, P.G. and Pardue, M.L. (1999) The two Drosophila telomeric transposable elements have very different patterns of transcription. Mol Cell Biol 19: 873–881. Deng, Z., Norseen, J., Wiedmer, A., Riethman, H. and Lieberman, P.M. (2009) TERRA RNA binding to TRF2 facilitates heterochromatin formation and ORC recruitment at telomeres. Mol Cell 35: 403–413. Díez, J., Rodriguez Vilariño, V., Medina, F. and Morcillo, G. (2006) Nucleolar localization of a reverse transcriptase related to telomere maintenance in Chironomus (Diptera). Histochem Cell Biol 126: 445–452. Ding, D., Zhou, J., Wang, M. and Cong, Y.-S. (2013) Implications of telomere-independent activities of telomerase reverse transcriptase in human cancer. FEBS J 280: 3205– 3211. Erkine, A.M., Magrogan, S.F., Sekinger, E.A. and Gross, D.S. (1999) Cooperative binding of heat shock factor to the yeast HSP82 promoter in vivo and in vitro. Mol Cell Biol 19: 1627– 1639. Farnung, B.O., Giulotto, E. and Azzalin, C.M. (2010) Promoting transcription of chromosome ends. Transcr 1: 140– 143. Fernandes, T., Madalena, C.R.G. and Gorab, E. (2012) Cloning and characterisation of a novel chromosome end repeat enriched with homopolymeric (dA)/(dT) DNA in Rhynchosciara americana (Diptera: Sciaridae). Chromosome Res. 20: 435–445.
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Telomeric transcriptome from Chironomus riparius Feuerhahn, S., Iglesias, N., Panza, A., Porro, A. and Lingner, J. (2010) TERRA biogenesis, turnover and implications for function. FEBS Lett 584: 3812–3818. Friedel, M., Nikolajewa, S., Sühnel, J. and Wilhelm, T. (2009) DiProGB: the dinucleotide properties genome browser. Bioinformatics 25: 2603–2604. Gonsalves, S.E., Moses, A.M., Razak, Z., Robert, F. and Westwood, J.T. (2011) Whole-genome analysis reveals that active heat shock factor binding sites are mostly associated with non-heat shock genes in Drosophila melanogaster. PLoS ONE 6: e15934. Gorab, E. (2003) Reverse transcriptase-related proteins in telomeres and in certain chromosomal loci of Rhynchosciara (Diptera: Sciaridae). Chromosoma 111: 445–454. Greenwood, J. and Cooper, J.P. (2012) Non-coding telomeric and subtelomeric transcripts are differentially regulated by telomeric and heterochromatin assembly factors in fission yeast. Nucleic Acids Res 40: 2956–2963. Guerrero, P.A. and Maggert, K.A. (2011) The CCCTC-binding factor (CTCF) of Drosophila contributes to the regulation of the ribosomal DNA and nucleolar stability. PLoS ONE 6: e16401. Hahn, S. (2004) Structure and mechanism of the RNA polymerase II transcription machinery. Nat Struct Mol Biol 11: 394– 403. Jolly, C., Metz, A., Govin, J., Vigneron, M., Turner, B.M., Khochbin, S. et al. (2004) Stress-induced transcription of satellite III repeats. J Cell Biol 164: 25–33. Juven-Gershon, T., Hsu, J.-Y., Theisen, J.W. and Kadonaga, J.T. (2008) The RNA polymerase II core promoter – the gateway to transcription. Curr Opin Cell Biol 20: 253–259. Kimura, R.H., Choudary, P.V., Stone, K.K. and Schmid, C.W. (2001) Stress induction of Bm1 RNA in silkworm larvae: SINEs, an unusual class of stress genes. Cell Stress Chaperones 6: 263–272. Kong, C.M., Lee, X.W. and Wang, X. (2013) Telomere shortening in human diseases. FEBS J 280: 3180–3193. Kramerov, D.A. and Vassetzky, N.S. (2011) SINEs. Wiley Interdiscip Rev RNA 2: 772–786. Kutach, A.K. and Kadonaga, J.T. (2000) The downstream promoter element DPE appears to be as widely used as the TATA box in Drosophila core promoters. Mol Cell Biol 20: 4754– 4764. Lakhotia, S.C. (2011) Forty years of the 93D puff of Drosophila melanogaster. J Biosci 36: 399–423. Li, T., Spearow, J., Rubin, C.M. and Schmid, C.W. (1999) Physiological stresses increase mouse short interspersed element (SINE) RNA expression in vivo. Gene 239: 367– 372. Ljungman, M. (2012) Transcriptional inhibition by DNA damage as a trigger for cell death. In The Cellular Response to the Genotoxic Insult The Question of Threshold for Genotoxic Carcinogens (Greim, H. and Albertini, R., eds), pp. 266–289. Royal Society of Chemistry, London. López, C.C., Nielsen, L. and Edström, J.E. (1996) Terminal long tandem repeats in chromosomes form Chironomus pallidivittatus. Mol Cell Biol 16: 3285–3290. López, C.C., Rodriguez, E., Díez, J.L., Edström, J. and Morcillo, G. (1999) Histochemical localization of reverse transcriptase in polytene chromosomes of chironomids. Chromosoma 108: 302–307.
© 2014 The Royal Entomological Society, 23, 367–380
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Luke, B., Panza, A., Redon, S., Iglesias, N., Li, Z. and Lingner, J. (2008) The Rat1p 5’ to 3’ exonuclease degrades telomeric repeat-containing RNA and promotes telomere elongation in Saccharomyces cerevisiae. Mol Cell 32: 465–477. Madalena, C.R.G. and Gorab, E. (2005) A chromosome end satellite of Rhynchosciara americana (Diptera: Sciaridae) resembling nematoceran telomeric repeats. Insect Mol Biol 14: 255–262. Madalena, C.R.G., Fernandes, T., Villasante, A. and Gorab, E. (2010) Curiously composite structures of a retrotransposon and a complex repeat associated with chromosome ends of Rhynchosciara americana (Diptera: Sciaridae). Chromosome Res 18: 587–598. Martinez, J.L., Edström, J.E., Morcillo, G. and Diez, J.L. (2001a) Telomeres in Chironomus thummi are characterized by different subfamilies of complex DNA repeats. Chromosoma 110: 221–227. Martinez, J.L., Sanchez-Elsner, T., Morcillo, G. and Diez, J.L. (2001b) Heat shock regulatory elements are present in telomeric repeats of Chironomus thummi. Nucleic Acids Res 29: 4760–4766. Martinez-Guitarte, J.L., Planelló, R. and Morcillo, G. (2007) Characterization and expression during development and under environmental stress of the genes encoding ribosomal proteins L11 and L13 in Chironomus riparius. Comp Biochem Physiol Part B 147: 590–596. Martínez-Guitarte, J.L., Díez, J.L. and Morcillo, G. (2008) Transcription and activation under environmental stress of the complex telomeric repeats of Chironomus thummi. Chromosome Res 16: 1085–1096. Martínez-Guitarte, J.-L., Planelló, R. and Morcillo, G. (2012) Overexpression of long non-coding RNAs following exposure to xenobiotics in the aquatic midge Chironomus riparius. Aquat Toxicol 110-111: 84–90. Mason, J.M., Frydrychova, R.C. and Biessmann, H. (2008) Drosophila telomeres: an exception providing new insights. Bioessays 30: 25–37. Maxwell, O.H., Belote, J.M. and Levis, R.W. (2006) Identification of multiple transcription initiation, polyadenylation, and splice sites in the Drosophila melanogaster TART family of telomeric retrotransposons. Nucleic Acids Res 34: 5498–5507. Mellor, J. (2010) Transcription: from regulatory ncRNA to incongruent redundancy. Genes Dev 24: 1449–1455. Morcillo, G., Santa-Cruz, M.C. and Díez, J.L. (1981) Temperature-induced Balbiani rings in Chironomus thummi. Chromosoma 83: 341–352. Nergadze, S.G., Farnung, B.O., Wischnewski, H., Khoriauli, L., Vitelli, V., Chawla, R. et al. (2009) CpG-island promoters drive transcription of human telomeres. RNA 15: 2186–2194. OECD (2001) Guideline for testing of chemicals, sediment-water chironomid toxicity test using spiked sediment. Orioli, A., Pascali, C., Pagano, A., Teichmann, M. and Dieci, G. (2012) RNA polymerase III transcription control elements: themes and variations. Gene 493: 185–194. Raha, D., Wang, Z., Moqtaderi, Z., Wu, L., Zhong, G., Gerstein, M. et al. (2010) Close association of RNA polymerase II and many transcription factors with Pol III genes. Proc Natl Acad Sci USA 107: 3639–3644. Rosén, M. and Edström, J. (2000) DNA structures common for chironomid telomeres terminating with complex repeats. Insect Mol Biol 9: 341–347.
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Santa-Cruz, M.C., Morcillo, G. and Diez, J.L. (1984) Ultrastructure of a temperature-induced Balbiani ring in Chironomus thummi. Biol Cell 52: 205–211. Schoeftner, S. and Blasco, M.A. (2008) Developmentally regulated transcription of mammalian telomeres by DNAdependent RNA polymerase II. Nat Cell Biol 10: 228–236. Shopland, L.S., Hirayoshi, K., Fernandes, M. and Lis, J.T. (1995) HSF access to heat shock elements in vivo depends critically on promoter architecture defined by GAGA factor, TFIID, and RNA polymerase II binding sites. Genes Dev 9: 2756–2769. Shpiz, S., Kwon, D., Rozovsky, Y. and Kalmykova, A. (2009) rasiRNA pathway controls antisense expression of Drosophila telomeric retrotransposons in the nucleus. Nucleic Acids Res 37: 268–278. Shpiz, S., Olovnikov, I., Sergeeva, A., Lavrov, S., Abramov, Y., Savitsky, M. et al. (2011) Mechanism of the piRNA-mediated silencing of Drosophila telomeric retrotransposons. Nucleic Acids Res 39: 8703–8711. Smale, S.T. and Kadonaga, J.T. (2003) The RNA polymerase II core promoter. Annu Rev Biochem 72: 449–479. Solovei, I., Gaginskaya, E.R. and Macgregor, H.C. (1994) The arrangement and transcription of telomere DNA sequences at the ends of lampbrush chromosomes of birds. Chromosome Res 2: 460–470. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731– 2739. Vrbsky, J., Akimcheva, S., Watson, J.M., Turner, T.L., Daxinger, L., Vyskot, B. et al. (2010) siRNA-mediated methylation of Arabidopsis telomeres. PLoS Genet 6: e1000986. Yamamoto, Y., Fujimoto, Y., Arai, R., Fujie, M., Usami, S. and Yamada, T. (2003) Retrotransposon-mediated restoration of
Chlorella telomeres: accumulation of Zepp retrotransposons at termini of newly formed minichromosomes. Nucleic Acids Res 31: 4646–4653. Yehezkel, S., Segev, Y., Viegas-Péquignot, E., Skorecki, K. and Selig, S. (2008) Hypomethylation of subtelomeric regions in ICF syndrome is associated with abnormally short telomeres and enhanced transcription from telomeric regions. Hum Mol Genet 17: 2776–2789. Zellinger, B. and Riha, K. (2007) Composition of plant telomeres. Biochim Biophys Acta 1769: 399–409.
Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: S1. MatInspector results for the three sequences. Matrix Family Library Version 8.2 (January 2010). Selected groups: Insects (0.75/Optimized). MatInspector Release professional 8.0.4, August 2010. (In these Tables: (+) strands = CriTER strand; (−) strands = αCriTER strand). S2. MatInspector results for the three sequences. Matrix Family Library Version 8.2 (January 2010). Selected groups: General Core Elements (0.75/Optimized). MatInspector Release professional 8.0.4, August 2010. Only appears some match in TsA sequences. (In these Tables: (+) strands = αCriTER strand; (−) strands = CriTER strand). S3. MatInspector results for the three sequences. Matrix Family Library Version 8.2 (January 2010). Selected groups: General Core Elements (0.75/Optimized – 0.10). MatInspector Release professional 8.0.4, August 2010. (In these Tables: (+) strands = αCriTER strand; (−) strands = CriTER strand). Explanation of scores from Genomatix programs [MatInspector from Genomatix Software GmbH 1988–2010 (Germany), Cartharius et al., 2005].
© 2014 The Royal Entomological Society, 23, 367–380
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Insect Molecular Biology (2014) 23(3), 367–380
doi: 10.1111/imb.12087
Telomeric transcriptome from Chironomus riparius (Diptera), a species with noncanonical telomeres
J. L. Martínez-Guitarte*‡, M. de la Fuente†‡ and G. Morcillo* *Grupo de Biología y Toxicología Ambiental. Facultad de Ciencias. Universidad Nacional de Educación a Distancia, UNED. Madrid. Spain; †Departamento de Ciencias y Técnicas Fisicoquímicas. Facultad de Ciencias. Universidad Nacional de Educación a Distancia, UNED. Madrid. Spain
scripts between control and stressful environmental conditions, supporting the idea that telomeric RNAs could have a relevant role in cellular metabolism in insect cells. Keywords: insect telomeres, non-telomerase telomere, non-coding RNA, heat shock, transcription regulation.
Abstract
Introduction
Although there are alternative telomere structures, most telomeres contain DNA arrays of short repeats (6–26 bp) maintained by telomerase. Like other diptera, Chironomus riparius has noncanonical telomeres and three subfamilies, TsA, TsB and TsC, of longer sequences (176 bp) are found at their chromosomal ends. Reverse transcription PCR was used to show that different RNAs are transcribed from these sequences. Only one strand from TsA sequences seems to render a noncoding RNA (named CriTER-A); transcripts from both TsB strands were found (CriTER-B and αCriTER-B) but no TsC transcripts were detected. Interestingly, these sequences showed a differential transcriptional response upon heat shock, and they were also differentially affected by inhibitors of RNA polymerase II and RNA polymerase III. A computer search for transcription factor binding sites revealed putative regulatory cis-elements within the transcribed sequence, reinforcing the experimental evidence which suggests that the telomeric repeat might function as a promoter. This work describes the telomeric transcriptome of an insect with nontelomerase telomeres, confirming the evolutionary conservation of telomere transcription. Our data reveal differences in the regulation of telomeric tran-
Telomeres are specialized nucleoprotein structures with repetitive DNA sequences that cap the ends of linear chromosomes. They are essential for completion of linear DNA replication, as well as for maintaining chromosome stability, and have a crucial role in regulating cellular senescence. Recent studies have also causally linked telomeres with many genetic, metabolic and inflammatory human diseases (Ding et al., 2013; Kong et al., 2013). Most eukaryotic telomeres are formed by simple and short repeats (6–26 bp) maintained by the enzyme telomerase, a reverse transcriptase. Nevertheless, a number of exceptions, with different types of telomeric DNA at their chromosome termini, have been described mainly in arthropoda and plants (Zellinger & Riha, 2007; Capkova Frydrychova et al., 2008). There are alternative telomere structures and alternative mechanisms to telomerase for telomere maintenance in insects (Biessmann & Mason, 2003). Among these, Chironomus (Diptera: Chironomidae) telomeres represent an interesting model as neither the short telomerase repeats (up to 30bp) nor the long LINE-like retrotransposons (HeT-A, TART, TAHRE, up to 4Kb), which are typical of Drosophila (Diptera: Drosophilidae) telomeres (Abad et al., 2004; Casacuberta & Pardue, 2005; Mason et al., 2008) are found. Instead, long arrays of DNA repeats, which are size (170–350 bp) and sequence species-specific, constitute the chromosome termini (Carmona et al., 1985; López et al., 1996, 1996; Rosén & Edström, 2000). These complex repetitive sequences must be functionally homologous to the other types of telomeric DNA and involved in the telomere-maintenance mechanism. The
First published online 18 February 2014. Corresponding author: José-Luis Martínez-Guitarte. Facultad de Ciencias. UNED. Senda del Rey 9. 28040 Madrid. Spain. Tel.: +34 91 398 7644; fax: +34 91 397 6697; e-mail: [email protected] ‡These authors contributed equally to this paper.
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localization of reverse transcriptase in Chironomid telomeres (López et al., 1999; Gorab, 2003; Díez et al., 2006) suggests that retrotranscription processes are also involved in the maintenance mechanism of these atypical telomeres. Curiously, composite structures of a retrotransposon and a complex repeat associated with chromosome ends have been found in Rhynchosciara [Diptera: Sciaridae (Madalena & Gorab, 2005; Madalena et al., 2010; Fernandes et al., 2012)]. Telomeres had been traditionally considered as transcriptionally inactive regions of chromosomes because of the lack of protein-coding genes in these regions, the heterochromatic state of the chromatin and the silencing of genes inserted into telomeric domains (telomere position effect). The discovery of telomeric repeat-containing RNAs (named TERRA) has challenged the view of transcriptionally silent telomeres (Azzalin et al., 2007; Schoeftner & Blasco, 2008) and redefined the telomere as a complex structure consisting not only of DNA and proteins but also RNA. As a result, research on telomere transcription has increased dramatically in recent years and the function of telomere transcripts is now the subject of intensive study, because of its possible role in the function of the telomere itself and the potential connections with aging and cancer (Caslini, 2010; Bah & Azzalin, 2012). TERRA RNA molecules have been reported in a number of eukaryotes with telomerase repeats, including mammals, birds, zebrafish, budding yeast, fission yeast and Arabidopsis (Solovei et al., 1994; Luke et al., 2008; Vrbsky et al., 2010; Bah & Azzalin, 2012; Bah et al., 2012; Greenwood & Cooper, 2012). The set of transcripts derived from telomeric and subtelomeric sequences has been defined as the telomeric transcriptome (Bah & Azzalin, 2012). In telomeres, transcription from both strands has been demonstrated only in fission yeast, where besides TERRA an anti-sense transcript (named ARIA) is produced together with sense and antisense subtelomeric transcripts (Bah et al., 2012; Greenwood & Cooper, 2012). The functions associated with the telomeric transcriptome still remains elusive. In humans and rodents a fraction of TERRA associates with telomeric heterochromatin and it has been proposed that it is involved in cancer progression, telomerase regulation, replicative senescence and epigenetic protection of telomeric DNA (Caslini, 2010). Telomeric transcriptional activity has also been detected in non-telomerase-dependent insect species. Drosophila telomeres, which are formed by non-long-terminal-repeat transposons, are in fact retrotranscribed to maintain the telomere length (Mason et al., 2008). Chironomus riparius, also known as Chironomus thummi, is the only species in which environmentally regulated telomeric transcription has been reported. Decades ago, it was established that its telomeres transcribe upon heat shock
(Morcillo et al., 1981). Interestingly, signals of telomere transcription, such as the existence of 350 nm ribonucleoprotein ethylenediaminetetraacetic acid (EDTA)positive particles and local labelling after in vivo [3H] uridine pulses, were clearly detected when telomeres were activated forming a giant puff under heat shock and other stressful conditions (Santa-Cruz et al., 1984; Barettino et al., 1988). Recently, using a high urea RNA extraction method, we detected the telomeric transcript by reverse transcription (RT)-PCR showing that it is a long noncoding RNA which is transcribed in a regular way and modulates its expression in response to different stimuli such as heat shock or specific chemicals, such as bisphenol A (Martínez-Guitarte et al., 2008, 2012). The telomeric RNA of C. riparius has been included in the group of noncoding RNAs as it does not show an open reading frame and in Northern blot it is detected as a ladder from 176 bp to >1000 bp (Martínez-Guitarte et al., 2008). The telomeres of this species show three variants of a 176 bp sequence, named TsA, TsB, and TsC, which differ in ∼10% of the sequence (Martinez et al., 2001a). We were able to show that TsA and TsB have a binding site for the heat shock factor, providing a molecular basis for telomere transcription upon heat shock (Martinez et al., 2001b), but the very nature of the transcripts remains unknown. In the present work, the telomeric transcriptome of C. riparius was analysed in an attempt to characterize the transcriptional activity in non-telomerase telomeres. Using RT-PCR with specific primers for the different telomeric sequences, the transcription profile was elucidated, finding three different transcripts of the telomere sequences. In addition, transcriptional behaviour during heat shock and the effects of different transcription inhibitors were analysed. The results support the idea that transcriptional activity in telomeres is under complex regulation and is altered under stressful environmental conditions. Bioinformatic analysis of telomeric sequences revealed putative transcription regulation sites within the telomeric repeats themselves, providing support for their different transcriptional behaviour. This work provides new insights into the telomeric transcriptome of a nontelomerase insect species and its complex regulation, thereby adding to the growing body of evidence regarding the existence of noncoding telomeric RNAs that might have a relevant role in cell metabolism. Results Analysis of Chironomus riparius telomeric RNA under control and heat shock conditions To analyse the variety of RNAs produced from the telomeres, specific primers were designed to retrotranscribe and detect by PCR each one of the strands of the three known telomeric sequences (TsA, TsB and TsC) © 2014 The Royal Entomological Society, 23, 367–380
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Table 1. Primers used in retrotranscription and in PCR. rpL13 (EF179386), TsA (M33211.1), TsB (AJ295633), and TsC (AJ295634) sequences from GenBank database were used to design the primers. The size is that obtained by reverse transcription-PCR in each case with the primers indicated Primer
Sequence
Method
Tel Retro M13 F Tel Retro B M13F L13 R L13 F L13 R2 M13 F RTPCR TsA R RTPCR TsB R RTPCR TsC R Tel RTPCR M13F B RTPCR TsA R B RTPCR TsB R B RTPCR TsC R B
5′-GTAAAACGACGGCCAGTAGATGCATCCAACG-3′ 5′-TGTTTTCCCAGTCACGACCGTTGGATGCATCT-3′ 5′-TTGGCATAATTGGTCCAG-3′ 5′-AACGTGCTTTCCCAAGAC-3′ 5′-TCATGAACTGGGAAGAGG-3′ 5′-GTAAAACGACGGCCAGT-3′ 5′-AAAATTTTCATGTTTTCG-3′ 5′-CACAGAATTGAAGTTTTTC-3′ 5′-AAAAGATTTCAACCTCTTC-3′ 5′-TGTTTTCCCAGTCACGAC-3′ 5′-CGAAAACATGAAAATTTT-3′ 5′-GAAAAACTTCAATTCTGTG-3′ 5′-GAAGAGGTTGAAATCTTTT-3′
RT RT RT PCR PCR PCR PCR PCR PCR PCR PCR PCR PCR
from C. riparius (Table 1). When RT-PCR was carried out, RNAs with primers specific for TsA and TsB sequences were detected (Fig. 1A) from only one strand for TsA, and from both strands for TsB; however, no signal was obtained with any of the specific primers for TsC sequences (data not shown). We named the RNAs retrotranscribed with Tel Retro M13 F primer as CriTER-A (Chironomus riparius TElomeric RNA from TsA) and CriTER-B, respectively, while the one observed when retrotranscription was carried out with Tel Retro B M13F was called αCriTER-B. The α indicates that the complementary strand of TsB was used as a template when producing this RNA. These results show that the telomeric transcriptome of C. riparius is formed by at least three different RNAs, produced from TsA and TsB sequences. Under normal growing conditions for larvae, the level of CriTER-A is much lower when compared with the RNAs transcribed from TsB sequences, which are much more abundant. Under environmentally stressful conditions, such as heat shock, CriTER-A transcripts dramatically increase. Treatment of fourth instar larvae for 2 h at 35 °C showed that CriTER-A levels increased fourfold in comparison with control larvae, whereas CriTER-B levels were only slightly increased, as were αCriTER-B RNA levels (Fig. 1B). No transcripts complementary to any of the TsC strands were found after the temperature shock (data not shown). These results show the differential transcriptional behaviour of the different telomeric repeats that, despite a rather similar sequence (∼10% of differences), revealed one strand of TsA as the main source for the telomeric RNA increase upon heat shock. TsB showed transcription in both strands, but with a lower activity in heat shock conditions. Effect of different RNA polymerase inhibitors on telomeric transcripts As RNA polymerase II and RNA polymerase III have been associated with the transcription of several long stress© 2014 The Royal Entomological Society, 23, 367–380
Size
262 bp 90 bp
119 bp
Figure 1. Telomeric RNAs and their transcription in heat shock conditions. (A) Detection of transcripts from TsA and TsB with specific primers. Total RNA was isolated and used to perform the retrotranscription with oligonucleotides to detect each strand. PCR was carried out with specific primers, and the transcripts were detected by electrophoresis in acrylamide gel. TsX + and TsX – notate strands retrotranscribed with Tel Retro M13 F and with Tel Retro B M13F oligonucleotides respectively. (B) Detection of CriTER-A, CriTER-B and αCriTER-B in control (C) and heat-shocked (HS) fourth instar larvae. The ribosomal protein L13 (rpL13) was used as reference gene. Gel images and quantification are shown. Values were normalized against rpL13. Means and standard deviations are shown from three independent experiments.
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responsive noncoding RNAs, we used different inhibitors to elucidate whether or not telomeric transcription and the different telomeric transcripts identified could be affected by them, both in control and heat shock conditions. Fourth instar larvae were in vivo exposed to the following: actynomicin D (AMD), a known general transcription inhibitor; 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), a specific inhibitor for RNA polymerase II; and (N-[1(3-(5-Chloro-3-methylbenzo[b]thiophen-2-yl-1-methyl-1Hpyrazol-5-yl)]-2-chlorobenzenesulfonamide), a specific inhibitor for RNA polymerase III (Bensaude, 2011; Ljungman, 2012). Figure 2A shows that, under normal growing conditions, AMD does not alter CriTER-A levels, but it decreases the levels of both transcripts of TsB. When heat shock-treated larvae were analysed, it was seen that AMD caused a general decrease of the three RNA levels. Yet, in a control situation, DRB only produced a decrease in CriTER-B levels, similar to that observed with AMD, whereas in heat shock-treated larvae, both RNAs showed a decrease in their levels, but to a lesser extent than with AMD. Levels of αCriTER-B were practically unaltered by the inhibitor, both in control conditions and in heat shocked larvae. Finally, with the Pol III inhibitor (Fig. 2B), CriTER-A, CriTER-B and αCriTER-B did not show any decrease in control larvae. Nevertheless, when heat-shocked larvae were analysed, a different pattern was observed. Although CriTER-A levels were not affected, CriTER-B and αCriTER-B levels suffered a sharp decline, especially strong for αCriTER-B (∼40%, as shown in Fig. 2C). The inhibition experiments suggest that RNA polymerase II is involved in the transcription of TsA and both strands of the TsB sequence, whereas RNA polymerase III has a relevant role in the synthesis of CriTER-B and αCriTER-B under heat shock conditions.
Computational analysis of promoters of the telomere sequence Our research group previously demonstrated that there is a heat shock element (HSE) in TsA and TsB sequences (Martinez et al., 2001b). This supports the hypothesis that regulation of telomeric transcription could depend on an internal promoter. Moreover, RNA polymerase inhibitors have shown that RNA polymerase II and RNA polymerase III could be involved in transcription at telomeres. To detect potential transcription factor binding sites related with Pol II and/or Pol III-type promoters, MATINSPECTOR software was used to predict the potential transcription factor binding sites in both strands of telomeric sequences. It is well established that presence of individual binding sites are not sufficient to indicate transcriptional function; however, comparative analysis of the putative elements appearing in each sequence provided
interesting results, which may be related to their differential transcriptional behaviour. For this analysis, CriTER-A and CriTER-B were the sense transcripts so they were considered plus strand, whereas the complementary one was considered to be antisense and then minus strand. A graphical summary of the more relevant results is presented in Figs 3 and 4. When default parameters were used, matches for core promoter elements were only found in TsA sequences (Supporting Information). The presence of a core promoter initiator element with high matrix similarity (0.952, where a perfect match would be 1.000) must be highlighted (Fig. 5). The most commonly occurring sequence motif in core promoters is probably Inr, which is recognized by TFIID, a transcription factor for Pol II, and this binding seems to be particularly important in the initial steps of transcription. It includes the transcription start site and is present in both TATA-containing and TATA-less promoters. In Drosophila promoters, its consensus sequence is TCAKTY, with the underlined A often being the +1 start site position (Kutach & Kadonaga, 2000; Smale & Kadonaga, 2003). If a lower matrix similarity threshold is used, other good matches for Inr elements can be observed (matrix similarity >0.80, Fig. 5), appearing in both strands of TsB but only in the TsA-plus strand. Curiously, these are the same strands that have been detected for producing RNA. Only one other significant core promoter element worth highlighting appeared in this analysis, this was a putative TATA box appropriately positioned (29 bp upstream of the transcription start site) in minus TsB. This strand had a resemblance in its architecture to a characteristic Pol II promoter (Fig. 3). The MATINSPECTOR program also predicted other worthwhile regulatory sequences by matching with insect matrix library. First, a HSE was detected in TsA and TsB (with matrix similarity up to 0.892), but not in the TsC sequence. Our group had previously demonstrated that it binds heat shock factor (HSF), a regulatory element associated with heat shock (Martinez et al., 2001b). Moreover, a putative recognition element for insulator factors boundary element-associated factor (BEAF)/DNA replication related element-binding factor (DREF) was predicted in both strands of TsA (matrix similarity up to 0.988). A manual motif search for consensus sequences of other relevant elements revealed a putative binding site for GAGA factor only in TsA-plus and a TC-box in both strands of TsA. This TC-box can bind a TBP-related factor specific to insects, called TRF1, which interacts with TFIIA and TFIIB and is also a component of TFIIIB (Smale & Kadonaga, 2003). As mentioned above, the different telomeric sequences of C. riparius are rather similar, with ∼90% similarity between them; however, the differences are not homogeneously distributed and, as shown in Fig. 3, there is one region © 2014 The Royal Entomological Society, 23, 367–380
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Figure 2. Effects of RNA synthesis inhibitors in control and heat shock conditions. Inhibitors of transcription reduce the RNA levels of telomeric transcripts. (A) Actinomycin D (AMD) and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) treatments were used to analyse the inhibition of the RNA polymerase II in RNA levels of CriTER-A, CriTER-B and αCriTER-B in control (C) and heat-shocked (HS) fourth instar larvae. (B) Changes for CriTER-A, CriTER-B and αCriTER-B levels in control (C) and heat shocked (HS) fourth instar larvae RNA levels in the presence of a specific RNA polymerase III inhibitor (RPIII inh). Values were normalized against ribosomal protein L13 (rpL13). Gel images and quantification are shown. Means and standard deviations are shown from three independent experiments. (C) Summary table with the decrease of RNA levels observed in each case as a percentage relative to the reference situation (control or heat shock).
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Figure 3. RNA polymerase II-related motifs in the telomeric sequences of Chironomus riparius. RNA polymerase II motifs are shown in the telomeric sequences and in a graphical map of each one. Location in plus (A) and minus (B) strands is shown. Vertical lines in the top of the sequences are for each 10 nucleotides, while the dots indicate the identity between the sequences. The last 17 bases of TsA (M33211.1), TsB (AJ295633), and TsC (AJ295634) sequences from the GenBank database were moved to the front to maintain heat shock element (HSE) together. Minus strands were obtained using the Sequence Manipulation Suite (SMS2, http://www.bioinformatics.org). BEAF, boundary element-associated factor; DREF, DNA replication related element-binding factor.
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Figure 4. RNA polymerase III-related motifs in the telomeric sequences of Chironomus riparius. RNA polymerase III motifs are shown in the telomeric sequences and in a graphical map of each one. Location in plus (A) and minus (B) strands is shown. Vertical lines in the top of the sequences are for each ten nucleotides, while the dots indicate the identity between the sequences. The last 17 bases of TsA (M33211.1), TsB (AJ295633) and TsC (AJ295634) sequences from GenBank database were moved to the front.
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Figure 5. Putative Inr elements found by MatInspector in the telomeric sequences. The different putative Inr elements are shown in a graph with their relative position to putative important sites in transcription by RNA polymerase II. The position of the putative Transcription start site is indicated as a bold A. In the table, the matrix similarity shows the probability of the Inr according to MatInspector. The position of each Inr is indicated in reference to sequences presented in Fig. 3: (+): plus strand, (−): minus strand.
∼30 bp towards the end of the plus strand that accumulates 50% of the dissimilarities. Two Inr in TsB are located in this region, but are absent in TsA because the differences observed turn them into a putative binding site for GAGA factor. It has been suggested that the GAGA factor acts as an anti-repressor by helping to disrupt nucleosomes, and it has also been related to heat shock genes and paused Pol II promoters (Shopland et al., 1995). Analysis of the other region that accumulates several differences showed a similar situation, in which the variations between the sequences lead to another distinctive
element in TsA, the recognition element for BEAF/DREF (predicted using the MATINSPECTOR program, with matrix similarity up to 0.988). The insulator factor BEAF seems to bind to DNA and regulates the expression of genes involving nucleosome-positioning conformations at promoters, which are coupled with RNA polymerase II pausing (Mellor, 2010; Gonsalves et al., 2011). Although there is a great diversity of RNA pol III promoters, there are two featured elements, boxes A and B, which bind the TFIIIC factor (Orioli et al., 2012). A manual search was carried out, which showed that both strands of © 2014 The Royal Entomological Society, 23, 367–380
Telomeric transcriptome from Chironomus riparius TsB and TsC have putative sites (Fig. 4). Yet, TsA presented putative A-boxes in both strands, whereas only its plus strand showed B-boxes. Regarding the termination signal formed by four T residues, as telomeric sequences of C. riparius are rich in AT pairs, there are several of these termination signals in all the sequences. The performed theoretical analysis showed that there are putative regulation elements in both TsA and TsB (Fig. 5), suggesting a possible molecular basis that could explain their transcription. In addition, this analysis suggested that the TsB-minus structure resembles a typical RNA Pol II promoter. Variations in key motif could be related to a different architecture in TsA, in which these motifs would be associated with clearing the region of nucleosomes to promote transcription (Mellor, 2010). Although RNAs from TsC or the minus strand of TsA have not been detected, the presence of putative binding sites opens up the possibility that they could be transcribed in other non-tested conditions. Discussion The present study reports the telomeric transcriptome of an insect with non-telomerase maintained telomeres. Three different RNAs, CriTER-A, CriTER-B and αCriTER-B, were identified in C. riparius that are transcribed from the telomeric DNA repeats TsA and TsB. The cellular pattern of telomeric transcripts differed in environmentally stressful conditions from that in the control situation. The levels of CriTER-A increased up to fourfold during heat shock. It is worth noting that, in contrast to TsA and TsB repeats, the TsC sequences did not appear to be transcribed. In addition, it was found that transcription inhibitors of RNA pol II and RNA pol III affect the set of transcripts derived from telomeric repeats. A bioinformatic analysis revealed the existence of putative promoter regulatory sequences within these transcribed repeats. These findings provide new insights into the telomeric RNA expression of C. riparius and open a new perspective of the transcriptional activity of telomeres, widening the field to those telomeres bearing telomerase-independent repeats. Long noncoding RNAs at eukaryotic telomeres are likely to be more prevalent than previously thought. The variety of telomeric RNAs found in C. riparius is in accordance with previous data, particularly from humans and yeasts, where the telomeric transcriptome has been thoroughly characterized and different transcripts from telomere sequences have been identified. In mammalian cells and budding yeast, TERRA transcripts contain G-rich telomeric repeats and tracks of adjacent subtelomeric sequences, whereas C-rich transcripts complementary to TERRA have not been detected (Azzalin et al., 2007; Luke et al., 2008; Schoeftner & Blasco, 2008). In addition, budding yeast contains ARRET RNA molecules, which are © 2014 The Royal Entomological Society, 23, 367–380
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complementary to the subtelomeric tract of TERRA but devoid of telomeric repeats (Luke et al., 2008). TERRA and ARRET have also been detected in Arabidopsis (Vrbsky et al., 2010). Furthermore, fission yeast telomeres also produce ARIA, a C-rich telomeric repeat-containing RNA, and α-ARRET that are subtelomeric transcripts complementary to ARRET (Bah et al., 2012; Greenwood & Cooper, 2012). In insects, only Drosophila telomeric transcriptome has been analysed in detail. Sense transcripts from the telomeric elements, HeT-A, TART and TAHRE, include open reading frames for proteins involved in their retrotransposition (Capkova Frydrychova et al., 2008); however, it has been described that TART and HeT-A are bidirectionally transcribed (Danilevskaya et al., 1999; Maxwell et al., 2006; Shpiz et al., 2009), showing a similar situation to mammals and yeasts. As in fission yeast, in C. riparius transcripts containing only telomeric repeats (ARIA) and transcripts derived from both strands have been identified, although in this latter case (yeast ARRET and α-ARRET) are derived from subtelomeric sequences. Although the transcript size for CriTER-A, CriTER-B and αCriTER-B corresponds to the telomeric monomer (176 bp), dimers, trimers and a smear denoting heterogeneous sizes and up to 10 Kb signals have also been previously detected for telomeric RNA in Northern blot after a high urea RNA extraction method (Martínez-Guitarte et al., 2008). The population of TERRA molecules is also very heterogeneous in length, ranging from ∼100 up to 9000 bases in mammals while it is shorter in yeast (0.80, searches with a decreased matrix similarity threshold were also performed to find more matches in this range. The DiProGB tool (Friedel et al., 2009) and MEGA software (Tamura et al., 2011) for motif search were also used to search for consensus sequences of specific core elements.
Funding This work was supported by Ministerio de Economia y Competitividad, Spain [grant CTM-2012-37547 from the Ciencias y Tecnologías Medioambientales programme].
Acknowledgements The authors wish to thank Dr T. Carretero (University of Zaragoza) and Ted Cater for critical reading of the manuscript.
Conflict of interest None declared.
References Abad, J.P., De Pablos, B., Osoegawa, K., De Jong, P.J., Martín-Gallardo, A. and Villasante, A. (2004) TAHRE, a novel telomeric retrotransposon from Drosophila melanogaster, reveals the origin of Drosophila telomeres. Mol Biol Evol 21: 1620–1624. Azzalin, C.M., Reichenbach, P., Khoriauli, L., Giulotto, E. and Lingner, J. (2007) Telomeric repeat containing RNA and RNA surveillance factors at mammalian chromosome ends. Science 318: 798–801. Bah, A. and Azzalin, C.M. (2012) The telomeric transcriptome: from fission yeast to mammals. Int J Biochem Cell Biol 44: 1055–1059. Bah, A., Wischnewski, H., Shchepachev, V. and Azzalin, C.M. (2012) The telomeric transcriptome of Schizosaccharomyces pombe. Nucleic Acids Res 40: 2995–3005. Balk, B., Maicher, A., Dees, M., Klermund, J., Luke-Glasser, S., Bender, K. et al. (2013) Telomeric RNA-DNA hybrids affect
telomere-length dynamics and senescence. Nat Struct Mol Biol 20: 1199–2205. Barettino, D., Morcillo, G. and Díez, J.L. (1988) Induction of the heat-shock response by carbon dioxide in Chironomus thummi. Cell Differ 23: 27–36. Bensaude, O. (2011) Inhibiting eukaryotic transcription: which compound to choose? How to evaluate its activity? Transcription 2: 103–108. Biessmann, H. and Mason, J.M. (2003) Telomerase-independent mechanisms of telomere elongation. Cell Mol Life Sci 60: 2325–2333. Capkova Frydrychova, R., Biessmann, H. and Mason, J.M. (2008) Regulation of telomere length in Drosophila. Cytogenet Genome Res 122: 356–364. Carmona, M.J., Morcillo, G., Galler, R., Martinez-Salas, E., Campa, A.G., Diez, J.L. et al. (1985) Cloning and molecular characterization of a telomeric sequence from a temperatureinduced Balbiani ring. Chromosoma 92: 108–115. Cartharius, K., Frech, K., Grote, K., Klocke, B., Haltmeier, M., Klingenhoff, A. et al. (2005) MatInspector and beyond: promoter analysis based on transcription factor binding sites. Bioinformatics 21: 2933–2942. Casacuberta, E. and Pardue, M.-L. (2005) HeT-A and TART, two Drosophila retrotransposons with a bona fide role in chromosome structure for more than 60 million years. Cytogenet Genome Res 110: 152–159. Caslini, C. (2010) Transcriptional regulation of telomeric noncoding RNA: implications on telomere biology, replicative senescence and cancer. RNA Biol 7: 18–22. Danilevskaya, O.N., Arkhipova, I.R., Traverse, K.L. and Pardue, M.L. (1997) Promoting in tandem: the promoter for telomere transposon HeT-A and implications for the evolution of retroviral LTRs. Cell 88: 647–655. Danilevskaya, O.N., Traverse, K.L., Hogan, N.C., DeBaryshe, P.G. and Pardue, M.L. (1999) The two Drosophila telomeric transposable elements have very different patterns of transcription. Mol Cell Biol 19: 873–881. Deng, Z., Norseen, J., Wiedmer, A., Riethman, H. and Lieberman, P.M. (2009) TERRA RNA binding to TRF2 facilitates heterochromatin formation and ORC recruitment at telomeres. Mol Cell 35: 403–413. Díez, J., Rodriguez Vilariño, V., Medina, F. and Morcillo, G. (2006) Nucleolar localization of a reverse transcriptase related to telomere maintenance in Chironomus (Diptera). Histochem Cell Biol 126: 445–452. Ding, D., Zhou, J., Wang, M. and Cong, Y.-S. (2013) Implications of telomere-independent activities of telomerase reverse transcriptase in human cancer. FEBS J 280: 3205– 3211. Erkine, A.M., Magrogan, S.F., Sekinger, E.A. and Gross, D.S. (1999) Cooperative binding of heat shock factor to the yeast HSP82 promoter in vivo and in vitro. Mol Cell Biol 19: 1627– 1639. Farnung, B.O., Giulotto, E. and Azzalin, C.M. (2010) Promoting transcription of chromosome ends. Transcr 1: 140– 143. Fernandes, T., Madalena, C.R.G. and Gorab, E. (2012) Cloning and characterisation of a novel chromosome end repeat enriched with homopolymeric (dA)/(dT) DNA in Rhynchosciara americana (Diptera: Sciaridae). Chromosome Res. 20: 435–445.
© 2014 The Royal Entomological Society, 23, 367–380
Telomeric transcriptome from Chironomus riparius Feuerhahn, S., Iglesias, N., Panza, A., Porro, A. and Lingner, J. (2010) TERRA biogenesis, turnover and implications for function. FEBS Lett 584: 3812–3818. Friedel, M., Nikolajewa, S., Sühnel, J. and Wilhelm, T. (2009) DiProGB: the dinucleotide properties genome browser. Bioinformatics 25: 2603–2604. Gonsalves, S.E., Moses, A.M., Razak, Z., Robert, F. and Westwood, J.T. (2011) Whole-genome analysis reveals that active heat shock factor binding sites are mostly associated with non-heat shock genes in Drosophila melanogaster. PLoS ONE 6: e15934. Gorab, E. (2003) Reverse transcriptase-related proteins in telomeres and in certain chromosomal loci of Rhynchosciara (Diptera: Sciaridae). Chromosoma 111: 445–454. Greenwood, J. and Cooper, J.P. (2012) Non-coding telomeric and subtelomeric transcripts are differentially regulated by telomeric and heterochromatin assembly factors in fission yeast. Nucleic Acids Res 40: 2956–2963. Guerrero, P.A. and Maggert, K.A. (2011) The CCCTC-binding factor (CTCF) of Drosophila contributes to the regulation of the ribosomal DNA and nucleolar stability. PLoS ONE 6: e16401. Hahn, S. (2004) Structure and mechanism of the RNA polymerase II transcription machinery. Nat Struct Mol Biol 11: 394– 403. Jolly, C., Metz, A., Govin, J., Vigneron, M., Turner, B.M., Khochbin, S. et al. (2004) Stress-induced transcription of satellite III repeats. J Cell Biol 164: 25–33. Juven-Gershon, T., Hsu, J.-Y., Theisen, J.W. and Kadonaga, J.T. (2008) The RNA polymerase II core promoter – the gateway to transcription. Curr Opin Cell Biol 20: 253–259. Kimura, R.H., Choudary, P.V., Stone, K.K. and Schmid, C.W. (2001) Stress induction of Bm1 RNA in silkworm larvae: SINEs, an unusual class of stress genes. Cell Stress Chaperones 6: 263–272. Kong, C.M., Lee, X.W. and Wang, X. (2013) Telomere shortening in human diseases. FEBS J 280: 3180–3193. Kramerov, D.A. and Vassetzky, N.S. (2011) SINEs. Wiley Interdiscip Rev RNA 2: 772–786. Kutach, A.K. and Kadonaga, J.T. (2000) The downstream promoter element DPE appears to be as widely used as the TATA box in Drosophila core promoters. Mol Cell Biol 20: 4754– 4764. Lakhotia, S.C. (2011) Forty years of the 93D puff of Drosophila melanogaster. J Biosci 36: 399–423. Li, T., Spearow, J., Rubin, C.M. and Schmid, C.W. (1999) Physiological stresses increase mouse short interspersed element (SINE) RNA expression in vivo. Gene 239: 367– 372. Ljungman, M. (2012) Transcriptional inhibition by DNA damage as a trigger for cell death. In The Cellular Response to the Genotoxic Insult The Question of Threshold for Genotoxic Carcinogens (Greim, H. and Albertini, R., eds), pp. 266–289. Royal Society of Chemistry, London. López, C.C., Nielsen, L. and Edström, J.E. (1996) Terminal long tandem repeats in chromosomes form Chironomus pallidivittatus. Mol Cell Biol 16: 3285–3290. López, C.C., Rodriguez, E., Díez, J.L., Edström, J. and Morcillo, G. (1999) Histochemical localization of reverse transcriptase in polytene chromosomes of chironomids. Chromosoma 108: 302–307.
© 2014 The Royal Entomological Society, 23, 367–380
379
Luke, B., Panza, A., Redon, S., Iglesias, N., Li, Z. and Lingner, J. (2008) The Rat1p 5’ to 3’ exonuclease degrades telomeric repeat-containing RNA and promotes telomere elongation in Saccharomyces cerevisiae. Mol Cell 32: 465–477. Madalena, C.R.G. and Gorab, E. (2005) A chromosome end satellite of Rhynchosciara americana (Diptera: Sciaridae) resembling nematoceran telomeric repeats. Insect Mol Biol 14: 255–262. Madalena, C.R.G., Fernandes, T., Villasante, A. and Gorab, E. (2010) Curiously composite structures of a retrotransposon and a complex repeat associated with chromosome ends of Rhynchosciara americana (Diptera: Sciaridae). Chromosome Res 18: 587–598. Martinez, J.L., Edström, J.E., Morcillo, G. and Diez, J.L. (2001a) Telomeres in Chironomus thummi are characterized by different subfamilies of complex DNA repeats. Chromosoma 110: 221–227. Martinez, J.L., Sanchez-Elsner, T., Morcillo, G. and Diez, J.L. (2001b) Heat shock regulatory elements are present in telomeric repeats of Chironomus thummi. Nucleic Acids Res 29: 4760–4766. Martinez-Guitarte, J.L., Planelló, R. and Morcillo, G. (2007) Characterization and expression during development and under environmental stress of the genes encoding ribosomal proteins L11 and L13 in Chironomus riparius. Comp Biochem Physiol Part B 147: 590–596. Martínez-Guitarte, J.L., Díez, J.L. and Morcillo, G. (2008) Transcription and activation under environmental stress of the complex telomeric repeats of Chironomus thummi. Chromosome Res 16: 1085–1096. Martínez-Guitarte, J.-L., Planelló, R. and Morcillo, G. (2012) Overexpression of long non-coding RNAs following exposure to xenobiotics in the aquatic midge Chironomus riparius. Aquat Toxicol 110-111: 84–90. Mason, J.M., Frydrychova, R.C. and Biessmann, H. (2008) Drosophila telomeres: an exception providing new insights. Bioessays 30: 25–37. Maxwell, O.H., Belote, J.M. and Levis, R.W. (2006) Identification of multiple transcription initiation, polyadenylation, and splice sites in the Drosophila melanogaster TART family of telomeric retrotransposons. Nucleic Acids Res 34: 5498–5507. Mellor, J. (2010) Transcription: from regulatory ncRNA to incongruent redundancy. Genes Dev 24: 1449–1455. Morcillo, G., Santa-Cruz, M.C. and Díez, J.L. (1981) Temperature-induced Balbiani rings in Chironomus thummi. Chromosoma 83: 341–352. Nergadze, S.G., Farnung, B.O., Wischnewski, H., Khoriauli, L., Vitelli, V., Chawla, R. et al. (2009) CpG-island promoters drive transcription of human telomeres. RNA 15: 2186–2194. OECD (2001) Guideline for testing of chemicals, sediment-water chironomid toxicity test using spiked sediment. Orioli, A., Pascali, C., Pagano, A., Teichmann, M. and Dieci, G. (2012) RNA polymerase III transcription control elements: themes and variations. Gene 493: 185–194. Raha, D., Wang, Z., Moqtaderi, Z., Wu, L., Zhong, G., Gerstein, M. et al. (2010) Close association of RNA polymerase II and many transcription factors with Pol III genes. Proc Natl Acad Sci USA 107: 3639–3644. Rosén, M. and Edström, J. (2000) DNA structures common for chironomid telomeres terminating with complex repeats. Insect Mol Biol 9: 341–347.
380
J. L. Martínez-Guitarte, M. de la Fuente and G. Morcillo
Santa-Cruz, M.C., Morcillo, G. and Diez, J.L. (1984) Ultrastructure of a temperature-induced Balbiani ring in Chironomus thummi. Biol Cell 52: 205–211. Schoeftner, S. and Blasco, M.A. (2008) Developmentally regulated transcription of mammalian telomeres by DNAdependent RNA polymerase II. Nat Cell Biol 10: 228–236. Shopland, L.S., Hirayoshi, K., Fernandes, M. and Lis, J.T. (1995) HSF access to heat shock elements in vivo depends critically on promoter architecture defined by GAGA factor, TFIID, and RNA polymerase II binding sites. Genes Dev 9: 2756–2769. Shpiz, S., Kwon, D., Rozovsky, Y. and Kalmykova, A. (2009) rasiRNA pathway controls antisense expression of Drosophila telomeric retrotransposons in the nucleus. Nucleic Acids Res 37: 268–278. Shpiz, S., Olovnikov, I., Sergeeva, A., Lavrov, S., Abramov, Y., Savitsky, M. et al. (2011) Mechanism of the piRNA-mediated silencing of Drosophila telomeric retrotransposons. Nucleic Acids Res 39: 8703–8711. Smale, S.T. and Kadonaga, J.T. (2003) The RNA polymerase II core promoter. Annu Rev Biochem 72: 449–479. Solovei, I., Gaginskaya, E.R. and Macgregor, H.C. (1994) The arrangement and transcription of telomere DNA sequences at the ends of lampbrush chromosomes of birds. Chromosome Res 2: 460–470. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731– 2739. Vrbsky, J., Akimcheva, S., Watson, J.M., Turner, T.L., Daxinger, L., Vyskot, B. et al. (2010) siRNA-mediated methylation of Arabidopsis telomeres. PLoS Genet 6: e1000986. Yamamoto, Y., Fujimoto, Y., Arai, R., Fujie, M., Usami, S. and Yamada, T. (2003) Retrotransposon-mediated restoration of
Chlorella telomeres: accumulation of Zepp retrotransposons at termini of newly formed minichromosomes. Nucleic Acids Res 31: 4646–4653. Yehezkel, S., Segev, Y., Viegas-Péquignot, E., Skorecki, K. and Selig, S. (2008) Hypomethylation of subtelomeric regions in ICF syndrome is associated with abnormally short telomeres and enhanced transcription from telomeric regions. Hum Mol Genet 17: 2776–2789. Zellinger, B. and Riha, K. (2007) Composition of plant telomeres. Biochim Biophys Acta 1769: 399–409.
Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: S1. MatInspector results for the three sequences. Matrix Family Library Version 8.2 (January 2010). Selected groups: Insects (0.75/Optimized). MatInspector Release professional 8.0.4, August 2010. (In these Tables: (+) strands = CriTER strand; (−) strands = αCriTER strand). S2. MatInspector results for the three sequences. Matrix Family Library Version 8.2 (January 2010). Selected groups: General Core Elements (0.75/Optimized). MatInspector Release professional 8.0.4, August 2010. Only appears some match in TsA sequences. (In these Tables: (+) strands = αCriTER strand; (−) strands = CriTER strand). S3. MatInspector results for the three sequences. Matrix Family Library Version 8.2 (January 2010). Selected groups: General Core Elements (0.75/Optimized – 0.10). MatInspector Release professional 8.0.4, August 2010. (In these Tables: (+) strands = αCriTER strand; (−) strands = CriTER strand). Explanation of scores from Genomatix programs [MatInspector from Genomatix Software GmbH 1988–2010 (Germany), Cartharius et al., 2005].
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