Molecular and Biochemical Parasitology, 52 (1992) 231-240 © 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00

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MOLBIO 01735

Transcript-specific developmental regulation of polyadenylation in Trypanosoma brucei mitochondria G. J a y a r a m a Bhat, A u g u s t i n e E. Souza, Jean E. F e a g i n a n d K e n n e t h S t u a r t Seattle Biomedical Research Institute, Seattle, WA, USA (Received 16 September 1991; accepted 2 January 1992)

Transcripts from many mitochondrial genes in kinetoplastids are heterogeneous in size, often occurring as 2 distinct size classes, but this cannot be accounted for by R N A editing alone. Analyses of transcripts from 6 mitochondrial genes of Trypanosoma brucei indicates that the size variation is due to poly(A) tail length. A larger fraction of CYb, COI and COIl transcripts have longer poly(A) tails in procyclic than in bloodstream forms. These transcripts are also more abundant in the procyclic forms. In contrast, a more substantial fraction of CR1 transcripts have longer poly(A) tails in bloodstream than in procyclic forms and these transcripts tend to be more abundant in bloodstream forms. Both ND4 and M U R F I transcripts show a similar size distribution of poly(A) tail lengths in these life cycle states although both transcripts are more abundant in bloodstream forms. Furthermore, genes with edited transcripts tend to have longer poly(A) tails than unedited transcripts. Transcript abundance is not strictly correlated with longer poly(A) tails. Thus, poly(A) length variation appears to be developmentally regulated in a transcript-specific fashion in T. brucei. This regulation of polyadenylation may influence mitochondrial gene expression as polyadenylation can regulate cytoplasmic gene expression in eukaryotes. Key words: Trypanosoma brucei; Polyadenylation; Mitochondrial gene transcript; Developmental regulation; Size variation

Introduction

African trypanosomes undergo dramatic shifts in respiratory metabolism during their life cycle. The bloodstream forms lack cytochromes and Krebs cycle enzymes and produce energy by glycolysis. Procyclic forms have cytochromes and a functional Krebs cycle and create energy primarily by cytochrome mediated respiration [1]. Vital components of the mitochondrial respiratory system are Correspondence address: Kenneth Stuart, Seattle Biomedical Research Institute, 4 Nickerson Street, Seattle, WA 98109, USA. Abbreviations: SDS, sodium dodecyl sulfate; CYb, apocytochrome b; MURFI, maxicircle unidentified open reading frame 1; MURF2, maxicircle unidentified open reading frame 2; NDI, N A D H dehydrogenase subunit 1; ND4, NADH dehydrogenase subunit 4; ND7, NADH dehydrogenase subunit 7; COI, cytochrome e oxidase subunit I; COIl, cytochrome e oxidase subunit II; COIII, cytochrome c oxidase subunit III; nt, nucleotides.

encoded in the mitochondrial genome and transcripts of all the mitochondrial genes are found in both stages of the life cycle [2-6]. Thus, the changes in energy metabolism probably entail regulation of mitochondrial gene expression. Transcripts from most mitochondrial genes of Trypanosoma brucei are heterogeneous in size with many occurring as 2 size classes that differ by 150-200 nt [2,4-6]. In some cases, the abundance of specific transcripts varies in a stage-specific manner and may affect one or both members of a set of transcripts from the same gene. Some kinetoplastid mitochondrial transcripts are also altered post-transcriptionally by the addition and occasionally the deletion of uridine residues from the primary transcript, a process termed RNA editing [79], but this is insufficient to account for the magnitude of size differences observed between transcript size classes [10]. Furthermore, transcripts for which editing is not reported also

232

exist in 2 size classes [6]. RNA and c D N A sequencing suggests that transcripts from the same gene have the same 5' ends [9,11-13]. Fewer 3' ends have been examined but the poly(A) addition sites for transcripts from the same gene appear to be at or near the same site [14,15]. Both size classes of transcripts are retained on oligo(dT) columns, indicating that they are polyadenylated, although more procyclic than bloodstream R N A is bound, implying that polyadenylation is differentially regulated during the life cycle [2,5,6,12] (unpublished results). Recently we have shown that the edited ATPase 6 m R N A from Leishmania tarentolae mitochondria also exists as two distinct size classes [11]. This R N A was examined by RNase H transcript mapping, and cloning and sequencing the poly(A) tail region, and the basis for the size difference was found to be due to variation in the length of the poly(A) tail [16]. Here, we report similar analyses of transcripts of 6 T. brucei genes. These results show that the length of the poly(A) tail is responsible for the observed differences between size classes and transcripts. The results also show that some mitochondrial transcripts preferentially have longer poly(A) tails in a life cycle stage where other mitochondrial transcripts have short poly(A) tails. This pattern is reversed in a different life cycle stage. Thus, the length of poly(A) tails is regulated in a transcript-specific fashion in the mitochondrion of T. brucei during development.

Materials and Methods

Cell culture and R N A isolation. T. brucei clone IsTaR 1 from stock E A T R O 164 was grown and isolated as described previously [17]. Bloodstream forms were harvested after 3 days of infection in rats. Cells were frozen in liquid nitrogen and stored at - 8 0 ° C prior to R N A extraction. R N A was isolated as described previously [2] and stored at - 8 0 ° C . Oligonucleotide primers. The following oligonucleotides were used in the present study.

Oligo(dT) (12-17 nt in length) was from Pharmacia. COI-1, 5'-CCAATCATTTTATGAGAAACACTTAAGCACACAAGACATAG-3'; COI-2, 5 ' - G G A A T T C C G T C A C A T G C T A A G C T A G A A T G - Y ; COI-3, 5'-CGGGATC C C G T G T T T C T C A T A A A A T G A T T G G - 3'; COII-FS, 5 ' - G C A A A C A A A A T T A T T T C A T TACACC-3'; CYb CS-2, 5'-CCGGATCCATA T A T T C T A T A T A A A C A A C C - 3 ' ; CYb-R, 5'CCTGACATTAAAAGACAACACAAATTTCTAAA-3'; ND4-1, 5'-CATATACATAATTGATTTCTATTCCAATACAAAAACTATAG-3'; M U R F 1-1, 5 ' - G C T T A G T A A T G T T A GTGTAGTATAATCACATAAGATAATAAAGCTGTAG-3'; CR1-3, 5'-CCGAATTCG T C A A A A T T T A A T T T C A C C G T G - 3 ' ; CR17, 5 ' - G G G A A T T C C T A A C A A T G G T T A A C TCAATGG-3' (underlined nucleotides denote cloning/linker sequence). CO1 PCR clones. An internal G + C - r i c h region of the COI transcript was cloned by reverse transcription and PCR amplification employing the COI-2 and COI-3 oligonucleotides, as described elsewhere [12]. Ten bloodstream form and 11 procyclic form clones were sequenced with Sequenase (U.S. Biochemical), according to the manufacturer's instructions. De-adenylation with RNase H and Northern blot analysis. RNase H digestion was done as described by Carrazana et al. [18]. Briefly, 10 ktg of total RNA was mixed with 250 ng of the appropriate oligonucleotide and incubated at 42°C for 10 min. The sample was then digested with 2.5 units of RNase H (BRL) in presence of 50 m M Tris-C1, pH 7.4/10 m M MgCI2/80 m M KCI/1 m M dithiothreitol at 37°C for 30 min, followed by ethanol precipitation of RNA. Northern blot analysis was done as described previously [11] using R N A ladders (BRL) as sizing markers. Hybridization with end-labeled oligonucleotides was carried out in 5 x SSPE (1 x SSPE = 90 m M NaC1/10 mM NaHzPO4/1 m M EDTA)/0.1% SDS at 60°C. Blots were washed 5 times with 5 x SSPE/ 0.1% SDS for 5 rain each at room temperature, followed by a final wash with 1 x SSPE/ 0.1% SDS for 1 min at 60°C and exposed to

233

film overnight. Riboprobe hybridization and washes were done according to Sambrook et al. [19], using the pTbCR1-4-55 c D N A which contains edited sequence.

--

B

P

+H

DB

P D

+H÷dT B P D

Results

Cytochrome oxidase (CO) I and II transcripts exhibit similar abundance patterns, with the small size class present in both life cycle stages and the larger present primarily in procyclic forms [4,5]. Northern blot analysis of total R N A probed with end-labeled COI-1 oligonucleotide shows a 1700-nt transcript in bloodstream forms, and 2 diffuse transcripts of 1700 and 1900 nt in procyclic forms (Fig. 1, three left-hand lanes), as seen previously [4,5]. Although they are less distinct in the photograph, the 2 bands are clearly visible in the autoradiogram. End-labeled COII-FS oligonucleotide detects a low abundance 750 nt transcript in bloodstream forms and a diffuse 900 nt transcript in procyclic forms (Fig. 2, three left-hand lanes); the smaller transcript is difficult to detect but its presence in both life cycle stages has been previously demonstrated [4]. None of these transcripts are detected in total R N A from a D K mutant that is devoid of mitochondrial D N A (Figs. 1 and 2, lanes D). Following RNase H digestion of R N A in the presence of oligo(dT), both the larger and smaller size classes of CO1 bands are replaced by a single size band that is slightly smaller than the small size class in both bloodstream and procyclic forms (Fig. 1, arrow, three righthand lanes). Both size classes are retained on oligo(dT) columns [5]. The longest continuous internal stretch of As in the CO1 gene is 4 nt, and this is insufficient to bind to an oligo(dT) column. Therefore, while accurate sizing of the small decrease in the small size class is difficult because of the relatively large size of the CO1 transcripts, it is likely that the 1700 nt transcript has a short (20-30 nt) poly(A) tail. The signal intensity compared to untreated R N A suggests that all CO1 transcripts have decreased to a single smaller size. Similar results are obtained with COIl; the larger size

5 w

3 w --COI-i

~_~

Fig. 1. Northern blot analysis of COI transcripts. 20 #g of total R N A from bloodstream forms (B), procyclic forms (P) or dyskinetoplastic mutants (D) with or without RNase H treatment and in the presence or absence of oligo(dT) were electrophoresed in formaldehyde/agarose gels, transferred to nylon membranes and hybridized to end-labeled COI-1. The 1900- and 1700-nt transcripts appear less distinct in the photograph but are clearly discernable in the autoradiogram. ( - ) indicates untreated samples. ( + H ) indicates R N A treated with RNase H alone as controls, and ( + H + d T ) indicates the total R N A treated with oligo(dT) and RNase H. Transcript sizes (in nt) are indicated on the left. The schematic map of the gene showing the location of COI-1 oligonucleotide is shown below the blot. The bar indicates 100 nt.

class transcript is not detected following oligo(dT)/RNase H treatment while the intensity of a smaller size class of transcript has increased (Fig. 2, three right-hand lanes), reflecting a decrease in size of the 900-nt COII transcript. Controls using RNase H treatment in the absence of oligo(dT) (Figs. 1 and 2, three middle lanes) resemble the untreated samples. As with COI transcripts, both size classes of COII transcripts are retained on oligo(dT) columns (unpublished results). The COII transcript has an internal A-

234

B P D

R-R-

~

900--

0

+H B P D

+l'l+dT B PD

750--

5'

I COII-FS

3' -,

r

Fig. 2. Northern blot analysis of COIl transcripts. Blots prepared as in Fig. 1 were hybridized to end-labeled oligonucleotide COIIFS. The transcript sizes are indicated to the left. ' R ' indicates non-specific hybridization to the rRNAs. The very small editing region of COII is shown by a vertical line. The bar indicates 100 nt.

rich region ( A A A A A T A A A A ) , about 150 nt downstream from the 5' end. If this region serves as a substrate in the oligo(dT)/RNase H experiment, the smaller size class should have decreased by 150 nt. However the decrease in size of smaller transcripts is barely detectable, thus it is likely that this internal region is not responsible for its binding to oligo(dT) column. Rather the binding is probably due to the presence of a short poly(A) tail. Thus, the length of the poly(A) tail is largely responsible for the size difference between the 2 size classes of transcripts. COII transcripts are edited by the addition of 4 uridines at the site of a genomic frameshift [20,21]. When the COII blot was reprobed with end-labeled COII-R (specific to edited transcripts), it detected only the 900-nt transcript in procyclic forms (data not shown) and no signal

was seen in bloodstream forms, confirming our earlier observation that editing in COIl transcripts is developmentally regulated [21]. This also suggests that edited COII transcripts have longer poly(A) tails than the unedited transcripts. The COI m R N A sequence has not been fully determined. The sequence of cDNAs produced by reverse transcription and PCR of the region of highest G vs. C strand bias (often associated with edited sequences) that represents about 25% of the m R N A shows no evidence of editing. There is also no editing at the 5' end and the predicted amino acid sequence is quite similar to that of L. tarentolae [22]. Taken together, these points suggest that COI m R N A is not edited. Unlike COII, therefore, the longer poly(A) tail of the larger size class of COI transcripts does not appear to correlate with editing of the transcript. Like COI and COII, the apocytochrome b (CYb) gene has two transcript size classes, 1200 and 1350 nt [2,10], both of which bind to oligo(dT) columns [2]. CYb transcripts are edited near the 5' end by the addition of 34 uridines which is insufficient to explain this 150 nt difference [6,10]. End-labeled CYb CS-2 oligonucleotide detects both edited and unedited transcripts and shows both size classes in similar abundance in bloodstream forms, and predominantly the larger transcripts in procyclic forms (Fig. 3A, three left-hand lanes). The pattern of CYb transcript abundance obtained in these experiments varies from that seen earlier [2,10]. This may reflect the proportion of slender versus stumpy forms, known to have differing patterns of CYb transcript abundance in bloodstream form RNA [3,6], or variations in the physiological conditions during growth. As with COI and COII, RNase H digestion of R N A in the presence of oligo(dT) resulted in the disappearance of the larger size class of CYb transcripts in both stages of the life cycle, with a concomitant increase in the smaller size class (Fig. 3A, three right-hand lanes) again indicating that polyadenylation is responsible for the size difference. The oligo(dT)/RNase H-treated bloodstream form sample contains some transcripts slightly smaller than the

235 B --

+H

B P D B P D

"I-H'PdT

-

B P D

BP

+H

0

+H.I-dT

B P D BP

D

R~

R1350-1200--

i!iiiii~iiiiii~i!:! ¸,.....

!

5'



3' -

CYb-R --

C~-CS2

Fig. 3. Northern blot analysis of CYb transcripts. Blots prepared as in Fig. 1 were hybridized to end-labeled CYb CS-2 (A) and end-labeled CYb-R (B). Transcript sizes are indicated to the left. The thicker region in the schematic diagram shows the edited region. 'R' indicates non-specific hybridization to the rRNAs. The bar indicates 100 nt.

B -

B

P D

÷H

B

+H+dT

P D

B

P

--

D

B

÷H

P

B

+H+CR1-3

P

B

P

R-

750--

750 520'

520-!iil

!ii~:i i

5 I

3 ~

CRI

-7

CRI

-3 q

Fig. 4. Northern blot analysis of CRI transcripts. Blots prepared as in Fig. 1 using oligo(dT) (A) or oligonucleotide CRI-3 (B) were hybridized to end-labeled CRI-7(A) or hybridized to an edited CRl-specific riboprobe (B). Transcript sizes are indicated to the left. The low intensity of the hybridization in panel A reflects the lower abundance of R N A edited through the CR1-7 region than edited at the 3' end. The similar intensity between bloodstream and procyclic form RNA is due to preferential detection of R N A edited at the 3' end. The thicker region in the schematic diagram shows the edited region. 'R' indicates the non-specific hybridization to ribosomal RNA. The bar indicates 100 nt.

procyclic RNA. This is consistent with the higher proportion of unedited transcripts in bloodstream forms which would be 34 nt

smaller than edited transcripts. The smaller size class of CYb transcripts contains predominantly unedited transcripts

236

while the larger size class contains edited transcripts [10,21]. When oligo(dT)/RNase Htreated samples were probed with end-labeled CYb-D oligonucleotide (specific for unedited transcripts), the 1200 nt transcript was detected in bloodstream form RNA (data not shown), confirming previous results. End-labeled CYbR, an oligonucleotide specific for edited transcripts, hybridized with 1350-nt transcripts in both bloodstream and procyclic forms for untreated and control RNase Htreated samples and with 1200-nt transcripts in the samples treated with both oligo(dT) and RNase H (Fig. 3B). These results indicate that the edited CYb transcripts have longer poly(A) tails than unedited transcripts, as is the case of COII transcripts. Recently we have observed that CR1 [9] transcripts are extensively edited with nearly 50% of the nucleotide sequence due to RNA editing [35]. The sizes of unedited and fully edited CR1 transcripts are 356 and 569 nt, respectively, excluding the poly(A) tail. Northern blot analysis of total RNA probed with an edited oligonucleotide CR1-7 (corresponds to a region approx. 150 nt from the 5' end) detected heterogeneously sized transcripts that range from 520 to 750 nt in bloodstream forms. In procyclic forms, both smaller as well as larger transcripts are barely detectable and appear unaltered from the bloodstream form pattern (Fig. 4A). The background hybridization seen in lanes B and P is also present in lane D (RNA from dyskinetoplastic mutant), and therefore may represent the cross-hybridization of CR1-7 to cytoplasmic ribosomal RNAs. When the RNA was probed with a riboprobe made from a cDNA specific to edited CR1 RNA, transcripts that range from 520-750 nt are abundantly detected in bloodstream forms, whereas predominantly 520-nt transcripts were detected in procyclic forms (Fig. 4B). It is likely that the 520-nt transcript represents nearly fully edited CR1 transcripts and that the smear is due to partially edited RNAs and editing in the poly(A) tails. The differences seen with respect to the quantity of CR1 transcript detected in Fig. 4A and B is probably due to the nature of the probes and

washing conditions. It is interesting to note that with the riboprobe (Fig. 4B), crosshybridization to cytoplasmic ribosomal RNAs is significantly reduced. We tested the possibility that the larger transcripts (smear above 520 nt) seen in bloodstream forms might have longer poly(A) tails by oligo(dT)/RNase H digestion. Probing the oligo(dT)/RNase H-treated RNA in a Northern blot with an edited oligonucleotide, CR1-7, resulted in diminished hybridization to larger transcripts and an increase in smaller sized transcripts (Fig. 4A, three right-hand lanes). The retention of some larger transcripts in oligo(dT)/RNase H-treated RNA may reflect the presence of inserted uridines in the poly(A) tail, interfering with the binding of oligo(dT). We also examined the size of CR1 transcripts after hybridization with oligonucleotide CR1-3, complementary to the most 3' 22 nt of the transcript, and treatment with RNase H. As shown in Fig. 4B (two right-hand lanes), the heterogeneous size of the larger transcripts is visibly reduced in bloodstream forms in CR1-3/RNase H-treated samples. These findings support the interpretation that the poly(A) tail is responsible for the observed size difference and that oligo(dT) hybridization is not able to effectively remove the complete poly(A) tail from all CR1 transcripts. This also suggests that poly(A) tail length is developmentally regulated for CR1 transcripts, being larger in bloodstream forms than in procyclic forms. Transcripts from two additional T. brucei mitochondrial genes were also examined. Both NADH dehydrogenase 4 (ND4) and maxicircle unidentified reading frame 1 (MURF1) encode 2 size classes of transcripts and both MURF1 transcripts are substantially more abundant in bloodstream than in procyclic forms [2,6,10]. Both have been examined for 5' editing and none has been detected [9] (unpublished results). The presence of complete open reading frames with homology to corresponding genes in other kinetoplastids [22] suggests there is little, if any, internal editing. End-labeled oligonucleotides were again used to probe Northern blots of

237

-B

PD

÷H B

P

-

+H + d T D

B

P

B

D

+H

P

D

B

p D

+H+dT B

p

D

1650-1450--

1600-1400--

5 w

3 #

-

5 e

NI~-I

3 t

MURFI

-

i

Fig. 5. Northern blot analysis of ND4 transcripts. Blots were prepared as in Fig. 1 and hybridized to end-labeled ND4-1. The transcript sizes are indicated to the left. The bar indicates 100 nt.

Fig. 6. Northern blot analysis of M U R F I transcripts. Blots were prepared as in Fig. 1 and hybridized to end-labeled M U R F I - I . The transcript sizes are indicated to the left. The bar indicates 100 nt.

untreated, RNase H-treated, and oligo(dT)/RNase H-treated RNAs. The ND4-1 probe detected heterogeneously sized transcripts in both stages of the life cycle, ranging in size from 1400 to 1600 nt (Fig. 5, three left-hand lanes). The M U R F I - 1 probe detected abundant transcripts between 1450 and 1650 nt in bloodstream forms and less abundant transcripts in the same size range in procyclic forms (Fig. 6, three left-hand lanes). In the presence of oligo(dT), the large and small size class transcripts of both ND4 and M U R F 1 are not detected and a single transcript is seen migrating approx. 20-30 nt below the smaller size class in both stages of the life cycle (Figs. 5 and 6, marked by arrows), indicating poly(A) tail length differences as the cause for the size variation.

Discussion

The results obtained in the present study strongly suggest that the size differences observed between two classes of transcripts for the 6 T. brucei genes examined are due to the presence of different lengths of poly(A) tail. In all cases, in the presence of oligo(dT) and RNase H, the larger size class decreases in size by about 150-200 nt after RNase H treatment. The smaller size class of COI, MURF1 and ND4 transcripts show shifts of approx. 20-30 nt, but such shifts are not detectable for CYb, COII and CR1 genes after oligo(dT)/RNase H treatment. However, the smaller size class from these genes binds to oligo(dT) columns [2] (unpublished results), suggesting that they have at least a small poly(A) tail. This is further substantiated by c D N A sequencing studies that reveal 2 size classes of ATPase 6 m R N A in L. tarentolae, one of which has an

238

approx. 18-25-nt poly(A) tail. The heterogeneous size of CR1 transcripts following RNase H/oligonucleotide treatment may be due to a problem in gel resolution or reflect uridines added in the poly(A) tail. The oligo(dT)/RNase H-treated samples for all genes examined in the present study are detected as relatively compact bands except CR1, regardless of the diffuse appearance of some of the untreated transcript bands. This suggests that the heterogeneous sizes of the transcripts are not artifactual, but are probably due to heterogeneous lengths of the poly(A) tail. Poly(A) tails of both edited and unedited transcripts in kinetoplastids contain uridines that are not coded in the gene and which appear to be added post-transcriptionally [14,15,20,23,24]. The number and location of the uridines varies in the poly(A) tails of different cDNAs from the same gene [14]. The data presented here cannot distinguish whether long and/or short poly(A) tails have added uridines, but the presence of these uridines may contribute to the size of the larger poly(A) tails. Possible regulation of U addition in poly(A) tails has not been examined. There are several recent reports of differences in the length of the poly(A) tail of individual mRNAs of a population of several mRNAs [25]. The changes in the polyadenylation status of these mRNAs have been correlated with the extent of polysome binding in vivo, particularly in developing systems, where rapid changes in the pattern of protein synthesis often occur [25]. The most dramatic example is the fate of 5 developmentally regulated mRNAs upon fertilization of Spisula oocytes [26]. Four of these transcripts are poly(A)- mRNAs that are translationally inactive in the oocyte. However, after fertilization, these mRNAs become polyadenylated and are recruited onto polysomes. The fifth transcript, a tubulin mRNA, is de-adenylated at fertilization, and is simultaneously excluded from the polysomes. Additional examples of modulation of poly(A) tail length accompanied with the changes in the translational efficiencies as a consequence of developmental or environmental stimuli have also been reported

[18,25-30]. The functional significance of the modulation of poly(A) tail length in the kinetoplastid mitochondrion is not known. However, its transcript specificity and stage specific regulation between bloodstream and procyclic forms may indicate a role in regulation of gene expression. The correspondence of efficient translation with larger poly(A) tails is particularly intriguing, as edited COII and CYb transcripts both have longer poly(A) tails than their unedited counterparts. The addition of long poly(A) tails and editing may only reflect that edited RNA will have existed longer than unedited RNA, thus having a greater opportunity for polyadenylation. However, both genes encode components of the mitochondrial respiratory system, which functions in procyclic forms, and both are edited predominantly in procyclic forms [10,21], implying a greater need to function in the life cycle stage having COII and CYb transcripts with longer tails. In contrast, edited CR1 transcripts are more abundant and have longer poly(A) tails in bloodstream forms. While the function of CR1 is not clear at this stage [35], it may be related to the NADH dehydrogenase complex, which may be preferentially employed in bloodstream forms [12]. The profound changes in the protein profiles of respiratory complexes during the differentiation from procyclic to bloodstream forms could be achieved in part by increasing or decreasing the efficiency of the translation which in turn could be effected by modulation of the poly(A) tail length. These changes may be sufficient to induce rapid changes in the protein synthetic pattern in the absence of significant m R N A synthesis or turnover [30]. The molecules with shorter poly(A) tails may be precursors to or products of those with longer poly(A) tails. In addition to its role in translation, the poly(A) tail in eukaryotic mRNAs is also implicated in stability, perhaps protecting them from 3' exonucleolytic attack [31]. Shortening of poly(A) tail length has been shown to precede m R N A degradation [31,32], and thus the mRNAs with short poly(A) tails observed in the present study may be molecules

239

generated during their degradation. Alternatively, the mRNAs with short poly(A) tails may be precursors of mRNAs with larger poly(A) tails, since polyadenylation is reported to take place in 2 steps in other organisms [33]. Kinetic analysis should prove useful to test these possibilities. Our recent studies on L. tarentolae ATPase 6 [16], and the results presented here on mitochondrial transcripts of T. brucei demonstrate the presence of different lengths of poly(A) tail in these transcripts. In addition, a greater proportion of procyclic than bloodstream form mitochondrial transcripts bind to oligo(dT) columns [6]. Together, these observations suggest that maxicircle transcripts are differentially polyadenylated. Stage-specific differential polyadenylation is seen in both edited and unedited transcripts. Other mitochondrial genes in kinetoplastids also have two size classes of transcripts, including the COIII, M U R F 2 and ND1 genes in T. brucei [4,24], the L. tarentolae CYb and COII genes [24,34], and the ND1, ND7, MURF2, COII, and COIII genes of Crithidia fasciculata [14,15,24]. It is likely that variation in the length of the poly(A) tail is responsible for the observed differences in size in these cases, as well. If poly(A) tail length influences m R N A function in these cases, then modulation of that length is likely to provide an additional level of posttranscriptional regulation in kinetoplastid mitochondria to cope with the varied environments encountered during the course of their life cycle. In this regard, this complex pattern of gene expression differs strikingly from that of other mitochondrial systems.

Acknowledgements We thank Dr. Peter J. Myler, Dr. Lys Guilbride, Dr. Robert Corell and other members of our laboratory for helpful discussions, and Peter Hickman for technical assistance. This work received support from NIH grants AI14102 and GM42188 to K.S., who is also a Burroughs Wellcome Scholar in Molecular Parasitology.

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Transcript-specific developmental regulation of polyadenylation in Trypanosoma brucei mitochondria.

Transcripts from many mitochondrial genes in kinetoplastids are heterogeneous in size, often occurring as 2 distinct size classes, but this cannot be ...
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