Plant Science 227 (2014) 90–100

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Plant Science journal homepage: www.elsevier.com/locate/plantsci

Review

HnRNP-like proteins as post-transcriptional regulators Wan-Chin Yeap a,b , Parameswari Namasivayam a , Chai-Ling Ho a,c,∗ a Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia b Sime Darby Technology Centre Sdn. Bhd., 1st Floor, Block B, UPM-MTDC Technology Centre III, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia c Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

a r t i c l e

i n f o

Article history: Received 4 April 2014 Received in revised form 17 July 2014 Accepted 18 July 2014 Available online 28 July 2014 Keywords: RNA-binding proteins Post-transcriptional regulation mRNA hnRNP-like proteins

a b s t r a c t Plant cells contain a diverse repertoire of RNA-binding proteins (RBPs) that coordinate a network of post-transcriptional regulation. RBPs govern diverse developmental processes by modulating the gene expression of specific transcripts. Recent gene annotation and RNA sequencing clearly showed that heterogeneous nuclear ribonucleoprotein (hnRNP)-like proteins which form a family of RBPs, are also expressed in higher plants and serve specific plant functions. In addition to their involvement in post-transcriptional regulation from mRNA capping to translation, they are also involved in telomere regulation, gene silencing and regulation in chloroplast. Here, we review the involvement of plant hnRNP-like proteins in post-transcription regulation of RNA processes and their functional roles in control of plant developmental processes especially plant-specific functions including flowering, chloroplastic-specific mRNA regulation, long-distance phloem transportation and plant responses to environmental stresses. © 2014 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Capping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternative splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Alternative splicing in response to stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Alternative splicing in circadian rhythm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Alternative splicing and flowering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Polyadenylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Trafficking of mRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. mRNA degradation and stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Initiation of translation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Telomere regulation and gene silencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Regulation in chloroplast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

91 92 93 94 94 94 96 96 97 97 98 98 99 99 99

∗ Corresponding author at: Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Tel.: +60 3 89467475. E-mail address: [email protected] (C.-L. Ho). http://dx.doi.org/10.1016/j.plantsci.2014.07.005 0168-9452/© 2014 Elsevier Ireland Ltd. All rights reserved.

W.-C. Yeap et al. / Plant Science 227 (2014) 90–100

1. Introduction RNA-binding proteins (RBPs) regulate the life cycle of messenger RNAs (mRNAs) and various aspects of post-transcriptional regulation. Eukaryotic cells contain a diverse repertoire of RBPs that orchestrate the RNA processes that begin in the nucleus and end in cytoplasm. RBPs govern the fate of transcripts by mediating mRNA processing from transcription of mRNA, as well as mRNA subcellular localization and translation to degradation. Some RBPs interact with small non-coding RNAs during DNA replication as well as regulation of transcription. Eukaryotic post-transcriptional RNA processes are accompanied by RBPs known as heterogeneous nuclear ribonucleoproteins (hnRNPs). Various lengths and sequences of precursor mRNAs (pre-mRNAs) are arranged in a sequence-specific arrangement with hnRNPs in messenger RNP (mRNP) complexes in the nucleus and the cytoplasm. HnRNPs are the predominant nuclear RBPs associated with various interacting proteins and pre-mRNA in multi-protein ribonucleoprotein complex throughout the processes including transcription, 5 capping, splicing, 3 cleavage, polyadenylation, mRNA export, translation, storage and turnover [1]. HnRNPs contain a prominent structure of RNA recognition motifs or K homology domains and auxiliary domains such as the glycine-rich motifs and the arginine–glycine–glycine box. RNA-recognition motifs and K homology domains are the major categories of ␣␤ proteins and the most common domains in plants (Fig. 1) [2]. The RNA-recognition motifs are composed of 80–90 amino acids that form a barrel-like topology of ␣-helices and ␤-sheet in the order ␤␣␤␤␣␤ while K homology domain consists of three stranded ␤-sheet packed against thee ␣-helices [1]. The majority of hnRNPs that are involved in transcription and also post-transcriptional processes have been

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functionally characterized in humans. Generally, hnRNPs are categorized into major and minor hnRNP groups. The major hnRNPs are composed of hnRNP A, hnRNP A/B, hnRNP C1/C2, hnRNP F, hnRNP H, hnRNP I/polypyrimidine-binding protein and hnRNP K/K homology domain protein while HuR, hnRNP A0 and CUG triplet repeat RNA binding protein 1 are the less abundant minor hnRNPs [1]. A complex set of hnRNP-like proteins are also expressed in plants. The Arabidopsis genome encodes 196 RNA-recognition motif-containing RBPs and 26 K homology domain containing proteins [2]. Among them, approximately 55 genes encode for hnRNP-like proteins. The proteome of developing rice seed alone contains 257 RBPs derived from 221 distinct genes in the rice genome [3–5]. A total of 194 RNA-recognition motifs containing RBPs and 24 K homology domains containing RBPs were identified in rice based on the functional annotation of the rice genome annotation project [6]. Only 19 of them are hnRNP-like proteins associated with prolamine mRNA in rice [4,5]. Plant hnRNP-like proteins are 45–60% identical to metazoan hnRNPs. Plant hnRNP-like proteins are highly conserved in their structural motif or domain arrangements and play multiple roles in post-transcriptional regulations as their metazoan counterparts. Nevertheless, they perform plant-specific biological functions including the regulation of floral development, circadian rhythms, hormone signalling, plant growth, response to stress, phloem transportation and regulation in chloroplast [7–13]. Plant hnRNP-like proteins shuttle between nucleus and cytoplasm as transporters of pre-mRNA and regulators of diverse RNA processing events. Plant hnRNP-like proteins are involved in the regulation of gene expression at the post-transcriptional level including pre-mRNA splicing, 5 capping, polyadenylation, RNA modification, transport from the nucleus to the cytoplasm,

Fig. 1. Schematic representation of modular structure arrangement and phylogenetic tree of heterogeneous nuclear ribonucleoprotein (hnRNPs)-like proteins in plants. The hnRNP-like proteins are classified based on the copies of RNA-recognition motifs and auxiliary domains. The RNA-recognition motifs comprised of 80–90 amino acids and two conserved regions, RNP1 and RNP2 interspersed throughout the motif. Other RNA-binding domains include the K homology (KH) domain, RGG (Arg-Gly-Gly) box, glycine-rich (G-rich), glutamine-rich (Q-rich), glycine–tyrosine rich (GY-rich), WW box, acidic, lysine–arginine–aspartic and glutamic acid rich (KRDE-rich) and M9 nucleocytoplasmic export signal (M9). These hnRNP-like proteins have been characterized in plants and are involved in multiple RNA processes including transcription, mRNA splicing, trafficking, stability, translation silencing and translation.

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Table 1 Plant hnRNP-like proteins in RNA metabolism and plant development. RBP

Accession number

Plant

Metazoan homologue

RNA metabolism

Molecular effects

References

SHI1 UBP1

Q8W4B1 AJ272011

Arabidopsis Tobacco

hnRNP K hnRNP A/B

Tobacco

hnRNP A/B

Cold responsive Heat response and stress granule NA

[15] [7,8,48,51]

Arabidopsis

hnRNP A/B

Alternative splicing

Wound tolerance and senescence

[9,10]

AtGRP7

AAD25815 AC007232 AAD25814 CAC00749 AAD12005 BAA97064 NP 179760

5 capping Alternative splicing mRNA stability mRNA turnover

Arabidopsis

hnRNP A1

Alternative splicing mRNA trafficking

[11,12,20–22,32,34,39–42]

AtGRP8

NP 195637

Arabidopsis

hnRNP A1

Alternative splicing

FCA

O04425

Arabidopsis

CUG-BP

OsFCA

Q6K271

Rice

CUG-BP

Flowering

[25]

HvFCA

FJ188402

Barley

CUG-BP

Seed germination

[26]

FLK HEN4

Arabidopsis Arabidopsis

hnRNP E hnRNP K

Floral transition Floral patterning

[27] [17]

PEPPER

NP 187112 AF525700 AF525701 NP 194330

Arabidopsis

hnRNP K

mRNA splicing Polyadenylation Gene silencing mRNA splicing Polyadenylation mRNA splicing Polyadenylation Alternative splicing Alternative splicing Polyadenylation Alternative splicing

Cold responsive; circadian clock; flowering and drought tolerance Cold responsive and circadian clock Floral transition

[28,29]

AtRNP1 RBP-A RBP-D CmRBP50 BTR1 StPTB StNova1 AKIP1

AJ303457 Q2R0P4 Q5Z655 G0ZS03 Q9LZ82 I1SWI9 XP 004239711 Q8LLE6

Arabidopsis Rice Rice Pumpkin Arabidopsis Potato Potato Broad bean

hnRNP A/B hnRNP A/B

Vegetative growth and pistil development Seed development Seed development Seed development Phloem translocation Virus- host interaction Phloem translocation Phloem translocation Stomata conductance

RBP47

Q0WW84

Arabidopsis

hnRNP A/B

[48]

28RNP cp29A cp29B cp31 cp33

P28644 Q08935 Q08937 X57079 X61113 Q04836

Spinach Tobacco

hnRNP A/B hnRNP-like

Heat response and stress granule Photosynthesis Photosynthesis

Arabidopsis

hnRNP-like

Photosynthesis

[13]

B3H7H5 Q0WQ48

Arabidopsis

PTB

[30,31,52]

STEP1

NP194208

Arabidopsis

hnRNP A1

Pollen germination, seed germination and flowering Cell death

NtGTBP1NtGTBP2 NtGTBP3

HM049166 HM049167 HM049168 Q9ASP6

Tobacco

hnRNP A1

Cell death

[56–58]

Arabidopsis

hnRNP-like

Cell identity during floral development

[59]

UBA1a

UBA1b

UBA1c

UBA2a

UBA2b

UBA2c

CP31A AtPTB1

LIF2

AtPTB2

PTB hnRNP K PTB hnRNP K hnRNP A/B or D

localization of mRNA, mRNA stability, turnover, and translation initiation (Table 1). Recently, characterization of plant specific hnRNP-like proteins has been conducted using various approaches to reveal the significance of these proteins in RNA metabolism in plants. In this review, we discuss the recent findings on the emerging roles of plant hnRNP-like proteins and their involvement in various aspects of post-transcriptional regulation in plants (Fig. 2). 2. Capping Eukaryotic nascent pre-mRNAs are protected from 5 –3 exonuclease degradation through a capping process. mRNA capping involves the cleavage of the 5 triphosphate of the pre-mRNA and addition of 7-methylguanosine (m7G) at the 5 -end of mRNA. The

RNA trafficking RNA localization RNA localization RNA trafficking RNA trafficking RNA trafficking RNA trafficking mRNA storage and stability mRNA storage and stability RNA stabilization mRNA stabilization mRNA degradation

mRNA stabilization mRNA degradation mRNA splicing Translation mRNA degradation Telomere length regulation Telomere length regulation Epigenetic modification

[8]

[21,22] [23–28,36,60–64]

[20] [5] [5] [43,44] [46] [45] [45] [16]

[65] [65–68]

[55]

capping process is initiated by phosphorylated carboxyl terminal domain of the largest subunit of RNA polymerase II during transcription. This terminal domain is phosphorylated on Ser-5 by cyclin-dependent kinase 7, a subunit of transcription factor IIH and dephosphorylated for recycling of RNA polymerase II [14]. Dephosphorylation of Ser-5 residue of carboxyl terminal domain in Arabidopsis is achieved by repressor protein FIERY2/carboxyl terminal domain phosphatase-like 1 (FRY2/CPL1) [14]. FRY2/CPL1 interacts with SHINYI (SHI1), a K homology domain protein in the repressor complex that modulates mRNA capping and also polyadenylation [15]. The shi1 mutant plants were generated by mutagenesis using ethyl methane sulfonate (EMS). They are resistant to abscisic acid in seed germination and sensitive to low temperature during vegetative growth than wild type,

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Fig. 2. The network of well-characterized plant hnRNP-like proteins involved in post-transcriptional regulation of mRNAs. Nascent nuclear pre-mRNAs are bound by hnRNP proteins in mRNP complexes and are then capped, spliced, cleaved and polyadenylated. Subsequently, nuclear mRNPs are exported to the cytoplasm accompanied by hnRNPs and other RBPs. Abnormal mRNAs are targeted to nonsense mediated decay while other mRNAs are bound by another set of hnRNP-like proteins and subjected to multiple fates in cytoplasm including localization, translation, storage and degradation. In response to environmental stimuli, translation initiation of several mRNAs is inhibited and these mRNAs are directed into large mRNP aggregates, stress granules or processing bodies for storage or degradation. RNA processes are indicated in red letters while plant hnRNP-like proteins are indicated in green letters.

resembling the phenotype of shi4 (a fry2/cpl1 allele) mutant plants, presumably due to disruption in the interaction between SHI1 and FRY2/CPL2 for mRNA capping of stress-responsive genes [15]. Loss-of-function mutations of both shi1 and shi4 resulted in changes in transcript expression levels of cold responsive genes, COR15A and COR47, increased mRNA capping efficiency, and altered polyadenylation site selection of stress-inducible genes [15]. The SHI1 and SHI4/FRY2/CPL1 proteins form a functional complex that represses expression of stress responsive genes under unstressed conditions in Arabidopsis through inhibition of transcription by preventing mRNA capping and transcription elongation [15]. The SHI1-SHI4/FRY2/CPL1 proteins may be replaced by the binding of activator components for activation of stress inducible genes in response to stress conditions in plants.

3. Alternative splicing Alternative splicing in plants increases transcriptome and proteome complexity. Widespread occurrence of alternative splicing enhances plant response to stress, circadian rhythms, floral induction, differentiation and growth [8–12,16–18]. Nuclear splicing of pre-mRNA and the selection of alternative splice sites are determined by the assembly of spliceosome containing small nuclear ribonucleoproteins (snRNPs) particles and serine/argininerich proteins (Fig. 3) [19]. The serine/arginine-rich proteins bind to specific regulatory elements – exonic splicing enhancers or intron splicing enhancers in pre-mRNA. They mediate protein interactions with U1 snRNP (at the 5 splice site) or U2AF (at the 3 splice site) (Fig. 3) [19]. The hnRNP-like family is another class of splicing

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Fig. 3. Splicing regulation is governed by cis-regulatory elements in the pre-mRNA and trans-acting splicing factors, serine/arginine rich protein (SR) and heterogeneous nuclear ribonucleoprotein (hnRNP). The splicing machinery, spliceosome is a macromolecular complex consists of five small nuclear ribonucleoproteins (snRNPs) (green circle) assembles on the exon after detection of splice sites around an exon. Serine/arginine-rich and hnRNP (orange circle) bind to the auxiliary sequences located at the exon or intron. The SR protein binds to the exon-splicing enhancer (ESE) sequences to enhance (solid green lines) splicing activity while hnRNP binds to the exon-splicing silencers (ESS) to repress splicing activity (dashed red lines). SR binds to ESE to recruit the U1 snRNP to the 5 -splice site, the U2AF to the 3 -splice site and the U2 snRNP to the branch point. When the hnRNP binds to ESS, it blocks the interaction of SR with other regulatory factors and promotes exon skipping, without affecting binding of U1 snRNP and U2AF to the 3 or 5 -splice sites. CBC, cap-binding complex; (Py)n , polypyrimidine-rich tract.

regulators that bind to pre-mRNA exon splicing silencers and block the binding site of splicing factors [19]. 3.1. Alternative splicing in response to stress Plant intronic and 3 -untranslated regions (3 -UTRs) of premRNAs are enriched with AU and U residues. These U residues are required for efficient intron processing or splice site selection. UBP1, a plant specific hnRNP-like protein from Nicotiana plumbaginifolia binds U-rich RNAs and nuclear polyA RNA to facilitate spliceosomal association. Transient overexpression of UBP1 in the protoplasts of N. plumbaginifolia enhanced splicing of inefficiently spliced introns of pre-mRNAs, which in turn increased the accumulation of the reporter mRNAs (with suboptimal introns or intronless) [7]. Further elucidation of the mechanism involved indicated that UBP1 interacts with the 3 -UTR of mRNAs that contain suboptimal introns or are intronless, and enhances accumulation of these mRNAs by preventing them from exonucleolytic degradation [7]. UBP1-associated protein 1a (UBA1a) and UBA2a proteins are two novel plant specific hnRNP-like proteins that interact with UBP1 protein in Arabidopsis [8]. Unlike UBP1, UBA1a and UBA2a are not involved in pre-mRNA splicing during transient overexpression of their genes in protoplasts [8]. UBA1a and UBA2a may be components of a complex that functions in other mRNA processes along with UBP1. Arabidopsis UBA2 is alternatively spliced at the 3 -UTR, giving rise to three variants known as UBA2a, UBA2b and UBA2c. They are inducible by mechanical wounding and are re-localized into the nucleus upon abscisic acid (ABA) treatment [9,10]. Constitutive overexpression of UBA2 resulted in cell death-like and yellow leaf phenotypes in Arabidopsis with elevated expression of genes associated with wounding, senescence, and defense [10]. The data indicated that the UBA2 protein may stabilize transcripts involved in the stress-induced senescence and cell death for efficient gene expression [10]. 3.2. Alternative splicing in circadian rhythm AtGRP7 is another member of the hnRNP-like superfamily involved in splicing. AtGRP7 is composed of an N-terminal RNArecognition motif and a long C-terminal tail rich in glycine residues. The glycine-rich C-terminal of AtGRP7 contains RGG box and a nuclear targeting sequence known as M9-like domain that is highly similar to human hnRNP A1 (Fig. 1) [20]. AtGRP7 influences the splicing of its own pre-mRNA and promotes alternative spliced AtGRP7 transcript that retains part of its intron, including a premature termination codon that is soon degraded in the nonsense-mediated decay pathway [12]. The AtGRP7 protein promotes the production of its short-lived transcript variants at the expense of mature mRNA with a cryptic 5 splice site [11,12]. This decreases the level of AtGRP7 proteins. This negative feedback loop

operates as a slave oscillator downstream of the circadian clock as temporal information within the cell. AtGRP7 also regulates the splicing of AtGRP8 (hnRNP-like protein) by repressing the oscillations of AtGRP8. AtGRP8 protein uses similar mechanism as AtGRP7 to generate alternatively spliced transcripts of its own with a premature termination codon [21]. Apart from autoregulation, AtGRP7 and AtGRP8 also regulate global alternative splicing of target transcripts in Arabidopsis, either by antagonizing or co-regulating SR proteins and cap-binding complex [22]. 3.3. Alternative splicing and flowering Several plant-specific RBPs have been characterized as flowering-time regulators in Arabidopsis, these proteins include flowering control locus A (FCA), flowering locus K (FLK), flowering locus Y (FY) and flowering time control protein A (FPA) (Fig. 4). FCA protein is the Arabidopsis putative homologue of human CUG triplet repeat RNA binding protein 1 which is a less abundant minor hnRNPs. FCA consists of two RNA-recognition motifs and a WW protein interaction domain (Fig. 1). Both FCA and FPA promote floral transition in flowering plants under long day condition by preventing the expression of FLOWERING LOCUS C (FLC), a MADS box transcription factor, which acts as a repressor of floral initiation and an integrator of autonomous and vernalization pathways (Fig. 4) [23]. FCA negatively regulates its own expression through alternative splicing giving rise to four FCA transcripts in ␣, ␤, ␦ and ␥ forms [18]. Only FCA-␥ among the variants encodes for the intronless full-length active FCA protein, which functions normally in the control of flowering time while FCA-␦ has a reading frame shift resulting in a codon that leads to premature termination [24]. Overexpression of FCA-␥ resulted in early flowering in Arabidopsis, a long-day plant. FCA and FY protein interactions are inhibited in the presence of ABA, resulting in a delay in plant flowering [23]. Alternative splicing and polyadenylation processes are conserved in FCA from dicot and monocot. Alternative splicing and polyadenylation also occur at intron 3 of OsFCA from rice resulted in ␣, ␤ and ␥ forms of OsFCA transcripts but not the ␦ form [25]. Overexpression of OsFCA partially rescued the late flowering phenotype of the Arabidopsis fca mutant without affecting FLC expression [25]. The N-terminal region of the OsFCA harbours a glycine-rich region, which is absent in Arabidopsis FCA [25]. Barley FCA also harbours a glycine-rich region at the N-terminus [26]. However, barley FCA did not rescue the late flowering phenotype of Arabidopsis fca mutant [26]. Despite the similarity in post-transcriptional regulation in monocot and dicot FCA, the additional protein domain may have contributed to variation in FCA function in plants. FLK (hnRNP-like protein) was implicated in the flowering time control for floral transition by repressing the expression of FLC [27]. FLK is a homologue of mammalian hnRNP E. Its function is independent of FCA and FPA in the autonomous pathway. FLK may

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Fig. 4. Schematic model of flowering time control regulation in Arabidopsis. Transition from vegetative phase to flowering phase in Arabidopsis is controlled by five major signaling pathways including vernalization, autonomous, photoperiod, aging and giberrellin (GA) pathways. The autonomous and vernalization pathways repress the activity of a MADS-box transcription factor and repressor of flowering, FLOWERING LOCUS C (FLC). The level of FLC mRNA is down-regulated by the autonomous pathway components, FCA, FY, FLK, FPA, AtGRP7, LD, FLD and FVE at the transcriptional, post-transcriptional and chromatin levels. CONSTANS (CO) in the photoperiod pathway stimulates the expression of the floral integrators, FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) while FLC suppresses their expression. LEAFY (LFY) which controls the floral meristem identity genes is regulated mainly by the giberrellin pathway. The aging pathway affects flowering through repression of flowering repressors and regulation of floral pathway integrators and meristem identity regulators.

interact with the gibberellin signaling pathway to promote and control flowering [27]. A close paralogue of FLK known as PEPPER is involved in vegetative growth and pistil development [28]. PEPPER also antagonizes FLK by positively regulating FLC [29]. Plants overexpressing PEPPER had a late-flowering phenotype and accumulated FLC transcripts [29]. It is possible that FLK represses FLC by downregulating PEP or HUA2. However, the mechanism of both FLK and PEP in transcriptional or post-transcriptional activation of FLC still remains unclear. Polypyrimidine tract-binding proteins have been highlighted as another key splicing factor in alternative splicing regulation of several key flowering regulators in Arabidopsis [30,31]. Three polypyrimidine tract-binding proteins homologues, AtPTB1, AtPTB2 and AtPTB3 from Arabidopsis generate two major splicing variants each. One encodes the full-length protein while the other variant contains a premature termination codon that is subjected to degradation by nonsense-mediated decay [30]. These AtPTB homologues are subject to extensive auto- and cross-regulation of their expression through alternative splicing coupled with nonsensemediated decay, akin to AtGRP7 and AtGRP8 [30]. Simultaneous knockdown of AtPTB1 and AtPTB2 triggered the retention of an intron within the 5 -UTR of the FLK mRNA that reduced the level of functional FLK protein and hence, should elevate the expression level of FLC [31]. Conversely, FLC expression was not detected in AtPTB1/AtPTB2 knockdowns and was strongly elevated in plants overexpressing AtPTB1/AtPTB2 [31]. The FLC transcript levels upon knockdown of AtPTB1/AtPTB2 was found to be suppressed by splice variant of FLOWERING LOCUS M (FLM), a member of the FLC-like clade that represses flowering [31]. Furthermore, upregulation

of AtPTB1 and AtPTB2 reduces exon skipping of PHYTOCHROME INTERACTING FACTOR 6. Alteration of alternative splicing of PHYTOCHROME INTERACTING FACTOR 6 transcripts upon knockdown of AtPTB1/AtPTB2 increases seed germination rates [31]. It is possible that polypyrimidine-binding protein-mediated alternative splicing events may affect diverse biological processes besides flowering and seed germination. In addition to the role of AtGRP7 in circadian clock, it is also involved in the control of flowering time. Transgenic plants ectopically overexpressing AtGRP7 had accelerated transition of flowering in short photoperiods with a reduction in FLC abundance [32]. The photoperiodic flower induction in Arabidopsis is mediated by endogenous circadian rhythm that favours earlier flowering in long photoperiods than short photoperiods [33]. These results suggested a link between circadian clock and floral transition regulation by AtGRP7. Furthermore, the expression levels of ent-copalyl diphosphate synthase (GA1) and ent-kaurene synthase (GA2) transcripts encoding enzymes involved in gibberellin biosynthesis were reduced in transgenic plants overexpressing AtGRP7 [34]. Gibberellins promote flowering in Arabidopsis through activation of the floral integrators SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 and LEAFY [35]. However, the plants overexpressing AtGRP7 had shorter vegetative stems, less leaves and early flowering, suggesting that AtGRP7 bypassed the effect of reduced gibberellin levels in floral promotion. HUA enhancer 4 (HEN4) is another K homology domain protein that interacts with HUA1 and HUA2 on stamen and carpel identity determination through regulation of AGAMOUS pre-mRNA, which is a floral homeotic gene that confers floral identity on meristems

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[17]. HEN4 transcripts are alternatively spliced in the generation of active HEN4 proteins. HEN4 together with HUA1 and HUA2 may facilitate efficient splicing or prevent alternative polyadenylation of AGAMOUS pre-mRNAs and promote AGAMOUS expression in Arabidopsis [17]. 4. Polyadenylation Once intronic sequences are removed during the splicing, cleavage and polyadenylation specific factor machineries will recognize the polyadenylation signals within the 3 -UTR of pre-mRNA. The transcript is cleaved and poly (A) polymerase adds a poly (A) tail to the 3 end of the RNA. The interlinking of splicing and polyadenylation affects the downstream events of mRNA including the mRNA export, stability, and also translation. In addition to the role of Arabidopsis FCA (hnRNP-like protein) in alternative splicing, it is also involved in the polyadenylation of its own transcripts. FCA negatively regulates its own expression by promoting the formation of its ␤ form transcripts through splicing and polyadenylation within intron 3 [18]. FCA-ˇ is the most abundant form of transcript that suppresses the formation of the functional ␥ form for temporal and spatial accumulation of functional FCA. The polyadenylation of FCA␤ within intron 3 requires the interaction of FCA with FY protein, which is a homologue of yeast polyadenylation factor Pfs2p [36]. In addition, FCA and FY regulate FLC pre-mRNA, possibly by promoting premature polyadenylation within FLC intron 1. The FLC intron 1 and its flanking exon sequences were sufficient for FCA dependent control [36]. However, the molecular mechanism determines whether FLC is directly or indirectly regulated by these hnRNPlike proteins remain to be ascertained. Similarly, floral homeotic gene AGAMOUS is also regulated by HEN4, HUA1 and HUA2 via efficient splicing or prevention of alternative polyadenylation. Plants with mutations in these three RBPs accumulate two long AGAMOUS transcripts as a result of alternative polyadenylation within the large second intron of AGAMOUS [17]. The primary defect of long AGAMOUS is undetermined, either premature polyadenylation or the inefficient splicing of the second intron, leads to utilization of cryptic polyadenylation signals in the second intron. 5. Trafficking of mRNAs Transcripts packaged into mRNP complexes with hnRNPs are transported across nuclear envelope from the nucleus to the cytoplasm by export factors. The hnRNPs interact with the export receptors on the nuclear pore complexes embedded in the nuclear envelope. Nucleo-cytoplasmic shuttling of hnRNPs has been extensively studied in yeast. The yeast nuclear abundant poly (A) RNA-binding protein 2 (Nab2), Npl3 and Nab4 proteins are hnRNPlike proteins required for export of mRNAs [37]. Npl3 protein assists the packaging of pre-mRNA into an export-competent RNP complex together with components of cytoplasmic filaments of nuclear pore complexes [38]. The mRNA export factors are released after translocation from the mature mRNPs and drive initiation of translation. Under stress conditions, hnRNP-like proteins facilitate the export of selective mRNAs encoding stress-related proteins from nucleus to the cytoplasm for translation and improve plant cellular recovery following stress. The Npl3 protein rapidly dissociates and terminates export of housekeeping mRNAs then re-localizes in the nucleus under heat shock conditions [38]. mRNAs encoding heat shock proteins are rapidly transcribed in the nucleus and exported to cytoplasm by Npl3, subsequently translated to cope with heat stress conditions without traffic hindrance at the nuclear pore by other housekeeping mRNAs [38]. Transportin 1 (AtTRN1), a member of the Arabidopsis importin ␤ family of nuclear transport receptors has been identified [20].

AtTRN1 interacts with human hnRNP A1 and hnRNP-like proteins including yeast Nab2p, AtRNP1, AtGRP7 and AtGRP8 proteins [20]. AtTRN1 interacts strongly with the glycine-rich C-terminus region of these proteins, which is highly similar to M9-like domain [20]. The M9-like domain is a nuclear import signal associated with nuclear import and mRNA export. It is also present in yeast Nab2p and human hnRNP A1 protein. Both AtRNP1 and AtGRP7 had nuclear import activity in a nuclear export signal competition assay [20]. AtGRP7 has RNA chaperone activity during cold adaptation in Escherichia coli, and bidirectionally shuttles between nucleus and cytoplasm using the reversibly photoswitchable fluorescent protein, DRONPA as protein shuttle monitoring tool [39,40]. Domain swapping and deletion experiments demonstrated that specific modular arrangement of N-terminal RNA-recognition motifs are structural determinants for RNA chaperone activity of AtGRP7 during cold adaptation in Arabidopsis [41]. Interestingly, overexpression of Arabidopsis AtGRP7 in rice enhanced stress tolerance and grain yield of rice under drought stress [42]. AtGRP7 harbours RNA chaperone activity and cold tolerance in Arabidopsis probably regulates the expression of different subset of stress-responsive genes related to drought tolerance in rice. Other hnRNP-like proteins, RBP-A, RBP-D and RBP-I from rice were identified and were specific for prolamine and glutelin mRNAs [5]. Prolamine and glutelin are rice seed storage proteins in developing rice seed endosperm that are transported via the actin cytoskeleton and are enriched on endoplasmic reticulum (ER) membranes. Both RBP-A and RBP-D were highly expressed during the rapid phase of storage protein accumulation. Their protein expression patterns were strongly correlated with major seed storage proteins [5]. RBP-A is co-localized with microtubules and cortical ER membranes whilst RBP-D was present in small particles closely associated with prolamine protein bodies and cytoskeletal elements on ER membranes [5]. The data suggested that RBP-A and RBP-D are involved in the localization of mRNAs encoding storage proteins to ER membranes and may also regulate their expression in the cytoplasm. Interestingly, plant cells also transport non-cell-autonomous RNAs through phloem via a long-distance trafficking mechanism involving a unique population of RBPs. The polypyrimidine tract-binding proteins are core proteins of the phloem-mobile RNP complexes utilized in the long-distance trafficking. A polypyrimidine-binding protein CmRBP50 from pumpkin is part of protein complex that mediate translocation of RNA [43]. Coimmunoprecipitation experiments indicated that the cmRBP50 RNP complex consists of phloem proteins and six transcripts that contain polypyrimidine tract-binding sequences (UUCUCUCUCUU) [43]. CmRBP50 may be involved in long-distance transport of transcription factors needed for the regulation of developmental or stress signalling events in plant tissues. Moreover, phosphorylation of serine residues at the C-terminus of CmRBP50 is required for effective and stable long-distance translocation of mRNA [44]. The phosphoserine residues of CmRBP50 are critical for interaction with phloem protein 16, GTP-binding protein and phosphoinositidespecific phospholipase-like protein in RNP complex assembly [44]. Additionally, the interaction of potato StPTB proteins with StNova1 suggests that K homology domain protein may also be involved in long-distance transport of mRNAs [45]. This is supported by the specific finding of a Nova-like BTR1 protein from Arabidopsis with the terminal regions of genomic RNA of tomato mosaic virus that contain regulatory elements for virus multiplication [46]. The overexpression study of BTR1 inhibited multiplication and cell-to-cell movement of tomato mosaic virus in Arabidopsis leaves [46]. This implies a negative regulation of K homology domain protein as a host factor that interacts with virus in inhibition of virus multiplication and local spread of virus in host plant.

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Fig. 5. Schematic model of mRNAs that are destined to stress granule (SG), processing body (PB) and translation initiation during stress conditions. Transcribed mRNAs form messenger ribonucleoprotein complexes (mRNPs) that are directed to translation or localized in two cytoplasmic mRNP granules – PB and SG depending on the environmental conditions. The PB contains components of mRNA decay for destruction of unnecessary mRNAs while SG contains translation initiation factors for temporarily storage of mRNAs that have been stalled during initiation of translation. The mRNAs can move between these SG and PB compartments and the mRNAs which entered PB can exit and re-initiate translation. UBP1 and RBP47 (orthologues of TIA-1 protein) are components of SG while AtPTBs are components of PB in plants. Other plant orthologous components of SG and PB such as TSN, TIAR, hnRNP E1, KH are yet to be identified in plants.

6. mRNA degradation and stability Interaction of the 3 -UTR in mRNAs with various parts of transcript including the 5 cap and poly (A)-tail determines the stability of the mRNA. The Vicia faba orthologue of Arabidopsis UBA2a, AAPKinteracting protein 1 (AKIP1) interacts with a guard-cell localized ABA-activated protein kinase (AAPK), which is involved in ABA signalling by controlling the aperture of stomatal pores and ion channels [16]. AKIP1 is another hnRNP-like protein with high similarity to mammalian hnRNP A/B and D proteins. ABA treatment induces the phosphorylation of AKIP1 by AAPK. The phosphorylated AKIP1 binds to mRNA encoding dehydrin, a ubiquitous stress-protective protein implicated in cell protection under stress conditions [16]. This results in dehydrin transcript stabilization. AKIP1 is constitutively expressed in the nucleus and rapidly relocalized into guard cell subnuclear structures resembling speckles in response to ABA, in a similar manner as UBA2a [16,47]. Nuclear speckles in mammalian systems are associated with splicing component storage, pre-spliceosome assembly and RNA processing [16]. This suggests a possible function of AKIP1 in mRNA storage, and the stability of ABA-responsive transcripts in response to ABA. The mRNA translation, degradation and temporary storage are dependent on environmental conditions and developmental stages of plants. Non-translated mRNAs are localized in two cytoplasmic mRNP granules; P-bodies or stress granules (Fig. 5) [48]. Stress granules contain translation initiation factors, poly (A)binding proteins and RBPs including Tudor-SN, TIA-1 and TIAR for storage and stabilization of non-translated mRNAs are arrested under stressed conditions [48]. Tudor-SN controls RNA stability during plant responses to environmental stresses while TIA-1 and TIAR proteins inhibit protein translation of target mRNAs through recruitment of untranslated mRNAs into stress granules that influence the survival of cells subjected to environmental stresses [49,50]. Studies have revealed that both stress granules and

P-bodies are synthesized in plants during heat stress. UBP1 and RBP47 are plant orthologues of TIA-1 protein that are components of stress granules [7]. UBP1 is involved in nuclear pre-mRNA maturation and prevents mRNA from degradation by binding to their 3 -UTR and inhibits 3 –5 exonucleolytic degradation [7]. Under unstressed conditions, UBP1 and RBP47 are localized in the nucleus. They are re-localized into cytoplasm together with translationally silenced housekeeping mRNAs in mRNPs and incorporated into heat stress granules under long-term heat-stress conditions [48]. The mutation of RNA-recognition motifs in UBP1 and RBP47 proteins inhibited formation of stress granules in plant cells, suggesting that these hnRNP-like proteins are core components of stress granules, which are required for stress granule assembly and also the storage of untranslated mRNAs under heat-stress condition. Recent immunoprecipitation study of Arabidopsis UBP1C mRNA complex indicated that UBP1C associates with mRNAs with U-rich 3 UTRs during normal condition and non-U-rich mRNAs during hypoxia [51]. These mRNAs were found to be related to hormone stimulus, membrane-associated proteins, cellulose synthase, cell wall, plasma membrane proteins, transcription factors, auxin regulators, small auxin-up response like proteins and cell cycle regulators [51]. This suggests that UBP1C, a component of stress granule is involved in stabilization, degradation or translation of stressinduced mRNAs during hypoxia.

7. Initiation of translation Gene expression can be modulated by the regulation of initiation of translation of mRNA. Structural features such as 5 -cap structure, poly (A) tail and internal ribosome entry sites within mRNAs are responsible for their translational fate. Translation initiation is stimulated by an interaction of poly (A)-binding protein with eIF4G1 and eIF4E, giving rise to a circular conformation, bridging

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Fig. 6. Translation initiation complex in eukaryotes. Initiation of translation involves the interaction of poly (A)-binding protein, eukaryotic initiation factors (eIF4G1 and eIF4E) and small ribosomal subunits to the 5 -end of an mRNA, giving rise to a circular conformation, bridging the 3 -UTR in close proximity to the 5 end of mRNA.

the 3 -UTR in close proximity to the 5 end of mRNA (Fig. 6) [52]. The mRNA circularization increases the efficiency of translation by promoting re-initiation of ribosomes on the same mRNA. An alternate way for circularization of mRNA has been discovered in higher eukaryotes, poly (A)-binding protein stimulates initiation of translation by binding to a poly (A)-binding protein interacting protein that resembles eIF4G1 [52]. Arabidopsis AtPTB1 and AtPTB2 are crucial for pollen germination. These proteins were proposed to be translation initiation factors of translationally repressed transcripts in mature pollen grains [52]. Moreover, AtPTBs is a component of P-body [30]. Pbodies contain components of mRNA decay machinery including the decapping enzymes and deadenylase that results in translational repression or degradation of unnecessary mRNAs [53]. AtPTBs may act as translation repressors that arrest the translation of target mRNA in P-bodies. In addition, translation is also controlled at late-initiation and post-initiation steps by hnRNP K and hnRNP E1 complex in mammalian cells. These complexes bind to a specific element in the 5 -UTR of mRNAs and inhibit the interaction of ribosomal subunit and pre-initiation complexes at the initiator AUG codon in mammalian cells [1]. It is possible that these complexes have a similar role in the regulation of differential translation in plants. However, plant orthologues of both hnRNP K and hnRNP E1 involved in translation regulation have yet to be characterized in higher plants. 8. Telomere regulation and gene silencing A unique group of hnRNP-like protein, telomere binding proteins are essential in controlling of telomere stability and the regulation of telomerase. Telomeres are specialized nucleoprotein complexes of chromosome ends, essential for chromosome stability and integrity. Human hnRNP A1 protein is directly involved in regulating telomere biogenesis and telomere length [54]. Arabidopsis STEP1 (single-stranded TBP 1) interacts with a single stranded telomeric repeat TTTAGGG, and inhibits telomerase extension through capping of chromosome ends which represses telomerase activity [55]. Moreover, three highly conserved plant G-strand specific telomere binding proteins (NtGTBP1, NtGTBP2 and NtGTBP3) homologous to human hnRNP A1 have been identified and characterized in tobacco [56]. The knockdown mutants of NtGTBP1 contained longer telomeres with extrachromosomal telomeric circles than the wildtype. They had severe developmental abnormalities due to genome instability [57]. NtGTBP1 is essential for regulation of the length of G-strand overhangs located at the 3 end of telomeres that protects telomeres against intertelomeric recombination [57,58]. HnRNP-like proteins are also implicated in RNA-mediated chromatin silencing at the locus encoding the major flowering repressor FLC through interaction with specific antisense transcripts. A

putative hnRNP-like protein member, LHP1 – Interacting Factor 2 (LIF2) is involved in polycomb complex-mediated control of gene expression. LIF2 interacts with a chromo domain protein LIKE HETEROCHROMATIN PROTEIN1 (LHP1), a subunit of plant polycomb repressive complexes in Arabidopsis [59]. LHP1 binds to multiple euchromatic genomic regions associated with an epigenetic silencing signal, H3K27me3. The loss-of-function of LIF2 plants had a mild-early flowering phenotype, with modified flowering time, floral developmental homeostasis and gynoecium growth determination [59]. LIF2 is involved in promoting expression of floral repressor FLC through epigenetic modifications and RNA processing mechanisms [59]. This suggests that LIF2 may modulate the activity of LHP1 at specific loci, either antagonizing or forming a partnership with LHP1 in response to environmental changes that control cell fate determination. The epigenetic control of FLC transcriptional silencing also involves both FCA and FPA [60,61]. In the autonomous pathway, FCA triggers chromatin silencing via targeted 3 processing at the proximal polyadenylation site in the antisense transcripts of FLC [61]. This triggers a histone demethylase activity that induces FLC transcriptional down-regulation of sense and antisense strands leading to FLC sense silencing [60,61]. The histone tails of FLC chromatin are deacetylated during vernalization. This converts FLC chromatin from an active form to heterochromatin-like state, leading to silencing of FLC [62]. FCA and FPA are also required for chromatin modification at loci subjected to siRNA-dependent chromatin silencing (AtMU1, AtSN1 and IG/LINE) that are involved in growth and development, independent of flowering time in plants [63]. Moreover, recent study in Arabidopsis suggested that FCA regulates the processing of primary transcripts of microRNA172 (miR172), miR398 and miR399 (temperature responsive miRNAs) in the thermosensory flowering pathway [64]. Thus, FCA and FPA proteins probably have an interplay between complex regulations of multiple RNA processes such as mRNA splicing, polyadenylation and chromatin modifications. They would do this through interactions with multiple RNA partners including non-protein coding RNAs for regulation of flowering.

9. Regulation in chloroplast Chloroplast gene expression is regulated by hnRNP-like proteins. Chloroplast ribonucleoproteins (cpRNPs) are hnRNP-like proteins involved in chloroplast RNA processing including 3 -end processing, RNA editing, and mRNA stability. Some chloroplast mRNAs contain an inverted repeat sequence in their 3 -UTR that confers a stable stem-loop structure for processing of 3 end and stabilization of 5 upstream region of mature mRNAs prior to translation in the chloroplast [65]. Rapid exonucleolytic degradation of mRNAs in chloroplast also can be achieved through the addition of poly (A) sequence to the endonucleolytic cleavage products of mRNA [66]. A 28RNP spinach protein is associated with 3 -end processing and 3 -UTR mediated RNA stabilization of chloroplast mRNAs, including the very high turnover psbA encoding the thylakoid D1 protein of photosystem II, rbcL encoding the large subunit of ribulose-1,5-bisphosphate carboxylase, petD encoding the subunit IV of cytochrome b6 /f complex and rps14 encoding the ribosomal protein S14 [65]. The depletion of 28RNP in chloroplasts interfered with the correct 3 -end processing of target mRNAs and resulted in a rapid degradation of target chloroplast mRNAs [65]. Another five cpRNPs: cp28, the homologue of 28RNP, cp29A, cp29B, cp31 and cp33 have been identified in tobacco. These cpRNPs have been implicated in the up-regulation of RNA processing, storage or translation in chloroplasts. However, depletion of all five cpRNPs from tobacco chloroplast extracts did not affect the 3 processing of petD

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mRNA whereas depletion of cp31 inhibited the editing of both psbL and ndhB mRNAs [65,67]. Multiple editing sites on chloroplast mRNAs were partially edited and the mRNAs were destabilized in CP31A Arabidopsis mutants [13]. Both CP31A and CP29A are required for stabilization of mRNAs under cold stress [68]. The CP31A associates with the 3 terminus of plastid NAD dehydrogenase (ndhF) and stabilizes ndhF transcript against 3 -exonucleolytic degradation [68]. 10. Conclusions and perspectives Genome-wide analyses in Arabidopsis and proteomic studies in rice have revealed a large number of RBPs. Plant hnRNP-like proteins are crucial for basic mechanisms in post-transcriptional regulation of gene expression. Hence their existence has been preserved during evolution in all eukaryotic lineages. Studies in plants have significantly indicated that plants express a complex set of plant hnRNP-like proteins that are critical components of post-transcriptional regulation of gene expression in higher plants. The functions of these proteins extend beyond the packaging of nascent RNA in RNP complexes to remarkably diverse roles in regulation of every aspect of RNA metabolism in plants. Many of these findings are derived from previous studies on plant hnRNPlike proteins in the control of flowering. Recent analyses have uncovered more plant hnRNP-like proteins involved in different processes and are likely to affect plant-specific functions including chloroplastic-specific regulation, long-distance phloem transport, and plant responses to environmental conditions apart from flowering. The advancement of genetics and genomics tools has increased our understanding of post-transcriptional regulation by hnRNPlike proteins in plants and their biological impact on plant growth and development. Their functions in mRNA splicing have been well studied in plants, yet their involvement in many mechanisms such as mRNA export, translation and gene silencing largely remain unknown or partially characterized. Limited plant hnRNP-like protein studies revealed their partners or mRNA targets involved. There is much to explore especially on the interaction between hnRNP-like proteins with other regulatory factors or their target mRNAs, the structural arrangements of these proteins in the RNP complex assemblies and the mechanisms involved. By combining high-throughput sequencing, genomic and proteomics approaches such as RNA immunoprecipitation, crosslinking immunoprecipitation, photoactivatable-ribonucleoside-enhanced crosslinking immunoprecipitation, should provide more comprehensive information on the structure and mechanism of RNP complexes comprising of plant hnRNP-like proteins, the co-factors and the target mRNAs that orchestrate a network of post-transcriptional regulatory mechanisms. In vivo approaches will further reveal physical interaction, RNA-binding specificity and mechanistic details of hnRNP-like proteins with RNA ligands in complex mRNPs in plant cells. Acknowledgements The authors thank Dr. Jonathan Gressel and the reviewers for their critical comments. References [1] G. Dreyfuss, M.J. Matunis, S. Pinol-Roma, C.G. Burd, HnRNP proteins and the biogenesis of mRNA, Annu. Rev. Biochem. 62 (1993) 289–321. [2] Z.J. Lorkovic, A. Barta, Survey and summary: genome analysis: RNA recognition motif (RRM) and K homology (KH) domain RNA-binding proteins from the flowering plant Arabidopsis thaliana, Nucleic Acids Res. 30 (2002) 623–635. [3] R.T. Morris, et al., RiceRBP: a database of experimentally identified RNA-binding proteins in Oryza sativa L., Plant Sci. 180 (2011) 204–211.

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