Polyadenylation Site Selection Research Articles

Comprehensive analysis of single-nucleotide variants and alternative polyadenylation between inbred and outbred pigs

Inbreeding can lead to the accumulation of homozygous single nucleotide polymorphisms (SNPs) in the genome, which can significantly affect gene expression and phenotype. In this study, we examined the impact of homozygous SNPs resulting from inbreeding on alternative polyadenylation (APA) site selection and the underlying genetic mechanisms using inbred Luchuan pigs. Genome resequencing revealed that inbreeding results in a high accumulation of homozygous SNPs within the pig genome. 3′ mRNA-seq on leg muscle, submandibular lymph node, and liver tissues was performed to identify differences in APA events between inbred and outbred Luchuan pigs. We revealed different tissue-specific APA usage caused by inbreeding, which were associated with different biological processes. Furthermore, we explored the role of polyadenylation signal (PAS) SNPs in APA regulation under inbreeding and identified key genes such as PUM1, SCARF1, RIPOR2, C1D, and LRRK2 that are involved in biological processes regulation. This study provides resources and sheds light on the impact of genomic homozygosity on APA regulation, offering insights into genetic characteristics and biological processes associated with inbreeding.

A post-transcriptional regulatory landscape of aging in the female mouse hippocampus.

Aging is associated with substantial physiological changes and constitutes a major risk factor for neurological disorders including dementia. Alterations in gene expression upon aging have been extensively studied; however, an in-depth characterization of post-transcriptional regulatory events remains elusive. Here, we profiled the age-related changes of the transcriptome and translatome in the female mouse hippocampus by RNA sequencing of total RNA and polysome preparations at four ages (3-, 6-, 12-, 20-month-old); and we implemented a variety of bioinformatics approaches to unravel alterations in transcript abundance, alternative splicing, and polyadenylation site selection. We observed mostly well-coordinated transcriptome and translatome expression signatures across age including upregulation of transcripts related to immune system processes and neuroinflammation, though transcripts encoding ribonucleoproteins or associated with mitochondrial functions, calcium signaling and the cell-cycle displayed substantial discordant profiles, suggesting translational control associated with age-related deficits in hippocampal-dependent behavior. By contrast, alternative splicing was less preserved, increased with age and was associated with distinct functionally-related transcripts encoding proteins acting at synapses/dendrites, RNA-binding proteins; thereby predicting regulatory roles for RBM3 and CIRBP. Only minor changes in polyadenylation site selection were identified, indicating pivotal 3'-end selection in young adults compared to older groups. Overall, our study provides a comprehensive resource of age-associated post-transcriptional regulatory events in the mouse hippocampus, enabling further examination of the molecular features underlying age-associated neurological diseases.

CRISPR-Cas9 mediated generation of a conditional poly(A) binding protein nuclear 1 (Pabpn1) mouse model reveals an essential role for hematopoietic stem cells

Poly(A) binding protein nuclear 1 (PABPN1) is known for its role in poly(A) tail addition and regulation of poly(A) tail length. In addition, it has been shown to be involved in alternative polyadenylation (APA). APA is a process regulating differential selection of polyadenylation sites, thereby influencing protein isoform expression and 3ʹ-UTR make-up. In this study, we generated an inducible Pabpn1flox/flox mouse model using crRNA-tracrRNA:Cas9 complexes targeting upstream and downstream genomic regions, respectively, in combination with a long single-stranded DNA (ssDNA) template. We performed extensive in vitro testing of various guide RNAs (gRNAs) to optimize recombination efficiency for in vivo application. Pabpn1flox/flox mice were generated and crossed to MxCre mice for validation experiments, allowing the induction of Cre expression in the bone marrow (BM) by poly(I:C) (pIC) injections. Validation experiments revealed successful deletion of Pabpn1 and absence of PABPN1 protein. Functionally, knockout (KO) of Pabpn1 led to a rapid and robust depletion of hematopoietic stem and progenitor cells (HSPCs) as well as myeloid cells, suggesting an essential role of Pabpn1 in the hematopoietic lineage. Overall, the mouse model allows an inducible in-depth in vivo analysis of the role of PABPN1 and APA regulation in different tissues and disease settings.

SRSF3 and SRSF7 modulate 3\u2032UTR length through suppression or activation of proximal polyadenylation sites and regulation of CFIm levels

BackgroundAlternative polyadenylation (APA) refers to the regulated selection of polyadenylation sites (PASs) in transcripts, which determines the length of their 3′ untranslated regions (3′UTRs). We have recently shown that SRSF3 and SRSF7, two closely related SR proteins, connect APA with mRNA export. The mechanism underlying APA regulation by SRSF3 and SRSF7 remained unknown.ResultsHere we combine iCLIP and 3′-end sequencing and find that SRSF3 and SRSF7 bind upstream of proximal PASs (pPASs), but they exert opposite effects on 3′UTR length. SRSF7 enhances pPAS usage in a concentration-dependent but splicing-independent manner by recruiting the cleavage factor FIP1, generating short 3′UTRs. Protein domains unique to SRSF7, which are absent from SRSF3, contribute to FIP1 recruitment. In contrast, SRSF3 promotes distal PAS (dPAS) usage and hence long 3′UTRs directly by counteracting SRSF7, but also indirectly by maintaining high levels of cleavage factor Im (CFIm) via alternative splicing. Upon SRSF3 depletion, CFIm levels decrease and 3′UTRs are shortened. The indirect SRSF3 targets are particularly sensitive to low CFIm levels, because here CFIm serves a dual function; it enhances dPAS and inhibits pPAS usage by binding immediately downstream and assembling unproductive cleavage complexes, which together promotes long 3′UTRs.ConclusionsWe demonstrate that SRSF3 and SRSF7 are direct modulators of pPAS usage and show how small differences in the domain architecture of SR proteins can confer opposite effects on pPAS regulation.

RNA chemical modification of m<sup>6</sup>A in plants

<p indent="0mm">N⁶-methyladenosine (m⁶A) is the most prevalent methylation modification form of eukaryotic messenger RNAs (mRNA). In plants, m⁶A participates in a variety of biological processes by affecting mRNA alternative polyadenylation, stability, and translation. In existing research, the absence of some methyltransferase complexes, such as methyltransferase A (MTA), methyltransferase B (MTB), FKBP12 interacting protein (FIP37), and virilizer (VIR), in plants causes embryonic lethality. Lowering gene expression levels of different methyltransferase components will cause severely hindered growth and development, such as growth retardation, over-proliferation of shoot apical meristem, aberrant gravity response, abnormal vascular tissue development, etc. Loss of demethylase AlkB homolog 10B (ALKBH10B) increases m⁶A modification levels of FLOWERING LOCUS T (<italic>FT</italic>), SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 3 (<italic>SPL3</italic>), and <italic>SPL9</italic>, lowering their stability, which leads to delayed flowering. The absence of AlkB homolog 9B (ALKBH9B) can increase the genomic m⁶A modification level of the alfalfa mosaic virus (AMV), weakening its infection capacity to plants. RNA binding proteins are mainly proteins with the YTH-domain. The evolutionarily conserved c-terminal region (ECT) represents main RNA binding proteins in plants. ECT2, ECT3, and ECT4, three kinds of ECTs, affect trichome branch numbers, precise leaf formation timing, and leaf morphology. Furthermore, the longer isoform of CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR 30 (CPSF30-L) which has mRNA binding function influences processes of polyadenylation site selection and phase separation. In addition to these features, these binding proteins are quite likely to be involved in plant response processes to adversity stress.<bold> </bold>Through the comprehensive analysis of existing studies, we considered that m⁶A is mainly enriched in the 3′ untranslated regions (3′UTR) region nearby the stop codon of plant mRNAs. As for the m⁶A modification enriched in the 5′ untranslated regions (5′UTR) region may be relevant to the higher abundance of chloroplast-related genes. The whole plant m⁶A-seq results show that RR[A]CH is a main m⁶A motif of plants. Sequencing of m⁶A at specific tissue sites or the sequence recognized by the binding protein indicated that UGU[A]Y is a plant-specific motif. As for the divergences in existing research about how m⁶A influences plant mRNA stability, we supposed that the existence of m⁶A is positively correlated with mRNA stability. However, there would be a negative correlation between m⁶A level and mRNA stability when m⁶A exists. In this article, we reviewed the research progress of plant m⁶A on three aspects, including m⁶A modification-related regulatory proteins and their functions, m⁶A position distribution and its motif characteristics, and the molecular mechanism of m⁶A regulation. Finally, we summarized the high-valued research emphasis and directions of m⁶A in plants.

Nudt21-mediated alternative polyadenylation of HMGA2 3'-UTR impairs stemness of human tendon stem cell.

Tendon-derived stem cells (TSCs) play a primary role in tendon physiology, pathology, as well as tendon repair and regeneration after injury. TSCs are often exposed to mechanical loading-related cellular stresses such as oxidative stress, resulting in loss of stemness and multipotent differentiation potential. Cytoprotective autophagy has previously been identified as an important mechanism to protect human TSCs (hTSCs) from oxidative stress induced impairments. In this study, we found that high-mobility AT-hook 2 (HMGA2) overexpression protects hTSCs against H2O2-induced loss of stemness through autophagy activation. Evidentially, H2O2 treatment increases the expression of Nudt21, a protein critical to polyadenylation site selection in alternative polyadenylation (APA) of mRNA transcripts. This leads to increased cleavage and polyadenylation of HMGA2 3′-UTR at the distal site, resulting in increased HMGA2 silencing by the microRNA let-7 and reduced HMGA2 expression. In conclusion, Nudt21-regulated APA of HMGA2 3′-UTR and subsequent HMGA2 downregulation mediates oxidative stress induced hTSC impairments.

The DEAD-box RNA helicase SHI2 functions in repression of salt-inducible genes and regulation of cold-inducible gene splicing.

Gene regulation is central for growth, development, and adaptation to environmental changes in all living organisms. Many genes are induced by environmental cues, and the expression of these inducible genes is often repressed under normal conditions. Here, we show that the SHINY2 (SHI2) gene is important for repressing salt-inducible genes and also plays a role in cold response. The shi2 mutant displayed hypersensitivity to cold, abscisic acid (ABA), and LiCl. Map-based cloning demonstrates that SHI2 encodes a DEAD- (Asp-Glu-Ala-Asp) box RNA helicase with similarity to a yeast splicing factor. Transcriptomic analysis of the shi2 mutant in response to cold revealed that the shi2 mutation decreased the number of cold-responsive genes and the magnitude of their response, and resulted in the mis-splicing of some cold-responsive genes. Under salt stress, however, the shi2 mutation increased the number of salt-responsive genes but had a negligible effect on mRNA splicing. Our results suggest that SHI2 is a component in a ready-for-transcription repressor complex important for gene repression under normal conditions, and for gene activation and transcription under stress conditions. In addition, SHI2 also serves as a splicing factor required for proper splicing of cold-responsive genes and affects 5' capping and polyadenylation site selection.

Epigenetic Regulation of mRNA Polyadenylation Site Selection.

The transcription of many genes is regulated through alternative splicing, with over 60% of genes in Arabidopsis ( Arabidopsis thaliana ) producing more than one mRNA ([Marquez et al., 2012][1]). The most common forms of alternative splicing are intron retention and the use of alternative

Modulation of Auxin Signaling and Development by Polyadenylation Machinery.

Polyadenylation influences gene expression by affecting mRNA stability, transport, and translatability. Here, we report that Cleavage stimulation Factor 77 (AtCstF77), a component of the pre-mRNA 3'-end polyadenylation machinery, affects polyadenylation site (PAS) selection in transcripts of some auxin signaling genes in Arabidopsis (Arabidopsis thaliana). Disruption of AtCstF77 reduced auxin sensitivity and decreased the expression of the auxin reporter DR5-GFP Null mutations of cstf77 caused severe developmental defects, but were not lethal as previously reported. cstf77-2 genetically interacted with transport inhibitor response 1 auxin signaling f-box 2 auxin receptor double mutants, further supporting that polyadenylation affects auxin signaling. AtCstF77 was ubiquitously expressed in embryos, seedlings, and adult plants. The AtCstF77 protein was localized in the nucleus, which is consistent with its function in pre-mRNA processing. We observed that PASs in transcripts from approximately 2,400 genes were shifted in the cstf77-2 mutant. Moreover, most of the PAS shifts were from proximal to distal sites. Auxin treatment also caused PAS shifts in transcripts from a small number of genes. Several auxin signaling or homeostasis genes had different PASs in their transcripts in the cstf77-2 mutant. The expression levels of AUXIN RESISTANT 2/INDOLE-3-ACETIC ACID 7 were significantly increased in the cstf77-2 mutant, which can partially account for the auxin resistance phenotype of this mutant. Our results demonstrate that AtCstF77 plays pleiotropic and critical roles in Arabidopsis development. Moreover, disruption of AtCstF64, another component of the polyadenylation machinery, led to developmental defects and reduced auxin response, similar to those of the cstf77-2 mutant. We conclude that AtCstF77 affects auxin responses, likely by controlling PAS selection of transcripts of some auxin signaling components.

Virulence- and signaling-associated genes display a preference for long 3'UTRs during rice infection and metabolic stress in the rice blast fungus.

Generation of mRNA isoforms by alternative polyadenylation (APA) and their involvement in regulation of fungal cellular processes, including virulence, remains elusive. Here, we investigated genome-wide polyadenylation site (PAS) selection in the rice blast fungus to understand how APA regulates pathogenicity. More than half of Magnaporthe oryzae transcripts undergo APA and show novel motifs in their PAS region. Transcripts with shorter 3'UTRs are more stable and abundant in polysomal fractions, suggesting they are being translated more efficiently. Importantly, rice colonization increases the use of distal PASs of pathogenicity genes, especially those participating in signalling pathways like 14-3-3B, whose long 3'UTR is required for infection. Cleavage factor I (CFI) Rbp35 regulates expression and distal PAS selection of virulence and signalling-associated genes, tRNAs and transposable elements, pointing its potential to drive genomic rearrangements and pathogen evolution. We propose a noncanonical PAS selection mechanism for Rbp35 that recognizes UGUAH, unlike humans, without CFI25. Our results showed that APA controls turnover and translation of transcripts involved in fungal growth and environmental adaptation. Furthermore, these data provide useful information for enhancing genome annotations and for cross-species comparisons of PASs and PAS usage within the fungal kingdom and the tree of life.

Abstract 4440: Differential regulation of p63 isoforms by alternative polyadenylation

Abstract p63, a transcription factor belonging to the p53 family, plays vital roles in epidermal development and tumorigenesis. p63 expresses from two different promoters generating two classes of proteins, TAp63 and ΔNp63 that differ in their N-terminus. Moreover, alternative splicing of the TA and ΔN isoforms in the C-terminus creates additional isoforms (α-ϵ); thus generating five TA and five ΔN p63 isoforms. The factors that give rise to this repertoire of p63 isoforms are not very well understood. Alternative polyadenylation (APA) can generate multiple isoforms of a gene with alternative 3' ends based on the polyadenylation site selection during cleavage and polyadenylation of mRNA. APA can give rise to isoforms that differ in the coding region (CDS APA) or that have varying lengths of 3' UTR (3' UTR APA). Recently, PABPN1 protein that binds the 3' poly(A) tail of mRNAs has emerged as a key regulator of APA. Here, we show that PABPN1 differentially regulates p63α and p63γ isoforms by alternative polyadenylation. Specifically, knockdown of PABPN1 increases the levels of p63γ mRNA as well as protein by regulating CDS APA of p63 gene. Moreover, knockdown of PABPN1 also generates p63α mRNAs with varying 3' UTR lengths. Although the mRNA levels of p63α remain unchanged upon PABPN1 KD, p63α protein is significantly decreased, suggesting that PABPN1 also regulates p63α protein translation. Interestingly, we also show that PABPN1 is a p63α target and that p63 transcriptionally regulates PABPN1 by binding to its promoter forming a negative feedback loop. Taken together, our study identifies PABPN1 as a novel regulator of various p63 isoforms by APA and that p63 in turn regulates PABPN1 transcriptionally. Citation Format: Shakur Mohibi, Jin Zhang, Xinbin Chen. Differential regulation of p63 isoforms by alternative polyadenylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4440.

Developmental dynamics of gene expression and alternative polyadenylation in the Caenorhabditis elegans germline

BackgroundThe 3′ untranslated regions (UTRs) of mRNAs play a major role in post-transcriptional regulation of gene expression. Selection of transcript cleavage and polyadenylation sites is a dynamic process that produces multiple transcript isoforms for the same gene within and across different cell types. Using LITE-Seq, a new quantitative method to capture transcript 3′ ends expressed in vivo, we have characterized sex- and cell type-specific transcriptome-wide changes in gene expression and 3′UTR diversity in Caenorhabditis elegans germline cells undergoing proliferation and differentiation.ResultsWe show that nearly half of germline transcripts are alternatively polyadenylated, that differential regulation of endogenous 3′UTR variants is common, and that alternative isoforms direct distinct spatiotemporal protein expression patterns in vivo. Dynamic expression profiling also reveals temporal regulation of X-linked gene expression, selective stabilization of transcripts, and strong evidence for a novel developmental program that promotes nucleolar dissolution in oocytes. We show that the RNA-binding protein NCL-1/Brat is a posttranscriptional regulator of numerous ribosome-related transcripts that acts through specific U-rich binding motifs to down-regulate mRNAs encoding ribosomal protein subunits, rRNA processing factors, and tRNA synthetases.ConclusionsThese results highlight the pervasive nature and functional potential of patterned gene and isoform expression during early animal development.

Abstract 865: Cytoplasmic polyadenylation element binding protein 2 splice variants have opposing functions in the epithelial to mesenchymal transition pathway

Abstract Alternative splicing of cytoplasmic polyadenylation element binding protein 2 (CPEB2) generates two isoforms, CPEB2B and CPEB2A, through inclusion/exclusion of exon 4, respectively, in mature mRNA. We previously reported that CPEB2B is essential to establish anoikis resistance (AnR), and promotes metastasis using in vivo orthotopic models. In contrast, CPEB2A decreases AnR and metastatic transformation. In the current studies, we examined the function of CPEB2 isoforms using next generation sequencing analysis of downstream signaling pathways. We found that CPEB2B expression upregulated AnR in breast cancer cells and induced key genes/proteins in the epithelial to mesenchymal transition (EMT) and hypoxic response (hypoxia inducible factor 1α (HIF1α)-dependent) pathways. Inhibition of either TWIST1 or HIF1α reduced the CPEB2B-mediated AnR in cells ectopically expressing CPEB2B, confirming a link between CPEB2 alternative splicing and the induction of the EMT/hypoxia pathways. We then extended our findings, and demonstrated that CPEB2 alternative splicing induced de novo translation of both HIF1α and TWIST1, whereas CPEB2A decreased translation of these factors. Furthermore, CPEB2 isoforms were found to modulate differential polyadenylation site selection of the TWIST1 3’UTR and competed for binding to HIF1α and TWIST1 3’UTR cytoplasmic polyadenylation element (CPE) site sequences. These results demonstrate that CPEB2A and CPEB2B modulate the translation of factors in the EMT/hypoxia pathways in an opposing fashion via differential polyadenylation site selection and subsequent translation. Citation Format: James T. DeLigio, Charles E. Chalfant, Margaret A. Park. Cytoplasmic polyadenylation element binding protein 2 splice variants have opposing functions in the epithelial to mesenchymal transition pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 865. doi:10.1158/1538-7445.AM2017-865

Arabidopsis thaliana microRNA162 level is posttranscriptionally regulated via splicing and polyadenylation site selection.

Arabidopsis microRNA162 (miRNA162) level regulation was studied under abiotic stresses, such as drought and salinity. The TaqMan® microRNA assay proved that A. thaliana miRNA162 level was elevated under these stresses, confirming its salt and drought responsiveness. The promoter region analyses of A. thaliana miRNA162a and b genes (MIR162a and MIR162b) identified numerous salinity and drought responsive elements. However, our results indicated that Arabidopsis MIR162a was presumably the main locus responsible for the mature ath-miRNA162 accumulation under the stresses tested, and the MIR162b was generally rather weakly expressed, both in control and under the stress conditions. The MIR162a structure was confirmed to be complex and the pri-miRNA162a hairpin structure was shown to span an alternative exon and an intron. The MIR162a transcription generated a few pri-miRNA162a splicing isoforms that could be functional and non-functional. Upon drought and salinity stresses, the regulation of the pri-miRNA162a alternative splicing pattern revealed an increase of a functional pri-miR162a isoform and a preferential distal polyA site selection under the stress conditions. Apart from the potential transcriptional regulation of the miRNA genes (MIRs) expression, the data obtained point to an essential role of posttranscriptional regulation of Arabidopsis microRNA162 level.

Polyadenylation site selection: linking transcription and RNA processing via a conserved carboxy-terminal domain (CTD)-interacting protein.

Despite the fact that the process of mRNA polyadenylation has been known for more than 40years, a detailed understating of the mechanism underlying polyadenylation site selection is still far from complete. As 3' end processing is intimately associated with RNA polymerase II (RNAPII) transcription, factors that can successively interact with the transcription machinery and recognize cis-acting sequences on the nascent pre-mRNA would be well suited to contribute to poly(A) site selection. Studies using the fission yeast Schizosaccharomyces pombe have recently identified Seb1, a protein that shares homology with Saccharomyces cerevisiae Nrd1 and human SCAF4/8, and that is critical for poly(A) site selection. Seb1 binds to the C-terminal domain (CTD) of RNAPII via a conserved CTD-interaction domain and recognizes specific sequence motifs clustered downstream of the polyadenylation site on the uncleaved pre-mRNA. In this short review, we summarize insights into Seb1-dependent poly(A) site selection and discuss some unanswered questions regarding its molecular mechanism and conservation.

The Nrd1-like protein Seb1 coordinates cotranscriptional 3' end processing and polyadenylation site selection.

Termination of RNA polymerase II (RNAPII) transcription is associated with RNA 3' end formation. For coding genes, termination is initiated by the cleavage/polyadenylation machinery. In contrast, a majority of noncoding transcription events in Saccharomyces cerevisiae does not rely on RNA cleavage for termination but instead terminates via a pathway that requires the Nrd1-Nab3-Sen1 (NNS) complex. Here we show that the Schizosaccharomyces pombe ortholog of Nrd1, Seb1, does not function in NNS-like termination but promotes polyadenylation site selection of coding and noncoding genes. We found that Seb1 associates with 3' end processing factors, is enriched at the 3' end of genes, and binds RNA motifs downstream from cleavage sites. Importantly, a deficiency in Seb1 resulted in widespread changes in 3' untranslated region (UTR) length as a consequence of increased alternative polyadenylation. Given that Seb1 levels affected the recruitment of conserved 3' end processing factors, our findings indicate that the conserved RNA-binding protein Seb1 cotranscriptionally controls alternative polyadenylation.

Reduced function of the RNA-binding protein FPA rescues a T-DNA insertion mutant in the Arabidopsis ZHOUPI gene by promoting transcriptional read-through.

T-DNA insertion mutants have been widely used to investigate plant gene functions. Unexpectedly, in several reported cases, the phenotype of T-DNA insertion mutations can be suppressed because of trans T-DNA interactions associated with epigenetic modification, which indicates that caution is needed when T-DNA mutants are used. In the present study, we characterized a novel process suppressing a T-DNA mutation. The spz2 (suppressor of zou 2) mutant was isolated as a suppressor of the phenotype of the zou-4 mutant caused by a T-DNA insertion in the first intron. The spz2 mutation partially recovered the native ZOU gene expression in the zou-4 background, but not in two other zou alleles, zou-2 and zou-3, with T-DNAs inserted in the exon and intron, respectively. The suppressed phenotype was inherited in a Mendelian fashion and is not associated with epigenetic modification. The recovery of the native ZOU gene expression in the spz2 zou-4 double mutant is caused by transcriptional read-through of the intronic T-DNA as a result of decreased proximal polyadenylation. SPZ2 encodes an RNA-binding protein, FPA, which is known to regulate polyadenylation site selection. This is the first example of FPA rescuing a T-DNA insertion mutation by affecting the polyadenylation site selection.

Seed Dormancy in Arabidopsis Is Controlled by Alternative Polyadenylation of DOG1.

DOG1 (Delay of Germination 1) is a key regulator of seed dormancy in Arabidopsis (Arabidopsis thaliana) and other plants. Interestingly, the C terminus of DOG1 is either absent or not conserved in many plant species. Here, we show that in Arabidopsis, DOG1 transcript is subject to alternative polyadenylation. In line with this, mutants in RNA 3' processing complex display weakened seed dormancy in parallel with defects in DOG1 proximal polyadenylation site selection, suggesting that the short DOG1 transcript is functional. This is corroborated by the finding that the proximally polyadenylated short DOG1 mRNA is translated in vivo and complements the dog1 mutant. In summary, our findings indicate that the short DOG1 protein isoform produced from the proximally polyadenylated DOG1 mRNA is a key player in the establishment of seed dormancy in Arabidopsis and characterizes a set of mutants in RNA 3' processing complex required for production of proximally polyadenylated functional DOG1 transcript.

The role of promoter cis-element, mRNA capping, and ROS in the repression and salt-inducible expression of AtSOT12 in Arabidopsis.

Inducible gene expression is a gene regulatory mechanism central to plant response to environmental cues. The inducible genes are often repressed under normal growth conditions while their expression levels are significantly elevated by conditions such as abiotic stresses. Induction of gene expression requires both cis-acting DNA elements and trans-acting proteins that are modulated through signal transduction pathways. Here we report several molecular events that affect salt induced expression of the Arabidopsis AtSOT12 gene. Promoter deletion analysis revealed that DNA elements residing in the 5′ UTR are required for the salt induced expression of AtSOT12. Cytosine methylation in the promoter was low and salt stress slightly increased the DNA methylation level, suggesting that DNA methylation may not contribute to AtSOT12 gene repression. Co-transcriptional processing of AtSOT12 mRNA including capping and polyadenylation site selection was also affected by salt stress. The percentage of capped mRNA increased by salt treatment, and the polyadenylation sites were significantly different before and after exposure to salt stress. The expression level of AtSOT12 under normal growth conditions was markedly higher in the oxi1 mutant defective of reactive oxygen species (ROS) signaling than in the wild type. Moreover, AtSOT12 transcript level was elevated by treatments with DPI and DMTU, two chemicals preventing ROS accumulation. These results suggest that repression of AtSOT12 expression may require physiological level of ROS and ROS signaling.

SR proteins control a complex network of RNA-processing events.

SR proteins are a well-conserved class of RNA-binding proteins that are essential for regulation of splice-site selection, and have also been implicated as key regulators during other stages of RNA metabolism. For many SR proteins, the complexity of the RNA targets and specificity of RNA-binding location are poorly understood. It is also unclear if general rules governing SR protein alternative pre-mRNA splicing (AS) regulation uncovered for individual SR proteins on few model genes, apply to the activity of all SR proteins on endogenous targets. Using RNA-seq, we characterize the global AS regulation of the eight Drosophila SR protein family members. We find that a majority of AS events are regulated by multiple SR proteins, and that all SR proteins can promote exon inclusion, but also exon skipping. Most coregulated targets exhibit cooperative regulation, but some AS events are antagonistically regulated. Additionally, we found that SR protein levels can affect alternative promoter choices and polyadenylation site selection, as well as overall transcript levels. Cross-linking and immunoprecipitation coupled with high-throughput sequencing (iCLIP-seq), reveals that SR proteins bind a distinct and functionally diverse class of RNAs, which includes several classes of noncoding RNAs, uncovering possible novel functions of the SR protein family. Finally, we find that SR proteins exhibit positional RNA binding around regulated AS events. Therefore, regulation of AS by the SR proteins is the result of combinatorial regulation by multiple SR protein family members on most endogenous targets, and SR proteins have a broader role in integrating multiple layers of gene expression regulation.