NTR 1 is required for transcription elongation checkpoints at alternative exons in Arabidopsis
2015; Springer Nature; Volume: 34; Issue: 4 Linguagem: Inglês
10.15252/embj.201489478
ISSN1460-2075
AutoresJakub Dolata, Yanwu Guo, Agnieszka Kołowerzo‐Lubnau, Dariusz Jan Smoliński, Grzegorz Brzyżek, Artur Jarmołowski, Szymon Świeżewski,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoArticle7 January 2015free access Source Data NTR1 is required for transcription elongation checkpoints at alternative exons in Arabidopsis Jakub Dolata Jakub Dolata Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznań, Poland Search for more papers by this author Yanwu Guo Corresponding Author Yanwu Guo Department of Protein Biosynthesis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Search for more papers by this author Agnieszka Kołowerzo Agnieszka Kołowerzo Department of Cell Biology, Faculty of Biology and Environment Protection, Toruń, Poland Centre for Modern Interdisciplinary Technologies, Nicolaus Copernicus University, Toruń, Poland Search for more papers by this author Dariusz Smoliński Dariusz Smoliński Department of Cell Biology, Faculty of Biology and Environment Protection, Toruń, Poland Centre for Modern Interdisciplinary Technologies, Nicolaus Copernicus University, Toruń, Poland Search for more papers by this author Grzegorz Brzyżek Grzegorz Brzyżek Department of Protein Biosynthesis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Search for more papers by this author Artur Jarmołowski Artur Jarmołowski Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznań, Poland Search for more papers by this author Szymon Świeżewski Corresponding Author Szymon Świeżewski Department of Protein Biosynthesis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Search for more papers by this author Jakub Dolata Jakub Dolata Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznań, Poland Search for more papers by this author Yanwu Guo Corresponding Author Yanwu Guo Department of Protein Biosynthesis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Search for more papers by this author Agnieszka Kołowerzo Agnieszka Kołowerzo Department of Cell Biology, Faculty of Biology and Environment Protection, Toruń, Poland Centre for Modern Interdisciplinary Technologies, Nicolaus Copernicus University, Toruń, Poland Search for more papers by this author Dariusz Smoliński Dariusz Smoliński Department of Cell Biology, Faculty of Biology and Environment Protection, Toruń, Poland Centre for Modern Interdisciplinary Technologies, Nicolaus Copernicus University, Toruń, Poland Search for more papers by this author Grzegorz Brzyżek Grzegorz Brzyżek Department of Protein Biosynthesis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Search for more papers by this author Artur Jarmołowski Artur Jarmołowski Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznań, Poland Search for more papers by this author Szymon Świeżewski Corresponding Author Szymon Świeżewski Department of Protein Biosynthesis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland Search for more papers by this author Author Information Jakub Dolata1,‡, Yanwu Guo 2,‡, Agnieszka Kołowerzo3,4, Dariusz Smoliński3,4, Grzegorz Brzyżek2, Artur Jarmołowski1 and Szymon Świeżewski 2 1Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznań, Poland 2Department of Protein Biosynthesis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland 3Department of Cell Biology, Faculty of Biology and Environment Protection, Toruń, Poland 4Centre for Modern Interdisciplinary Technologies, Nicolaus Copernicus University, Toruń, Poland ‡These authors contributed equally to this work *Corresponding author. Tel: +48 22 5925722; E-mail: [email protected] author. Tel: +48 22 5925725; E-mail: [email protected] The EMBO Journal (2015)34:544-558https://doi.org/10.15252/embj.201489478 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The interconnection between transcription and splicing is a subject of intense study. We report that Arabidopsis homologue of spliceosome disassembly factor NTR1 is required for correct expression and splicing of DOG1, a regulator of seed dormancy. Global splicing analysis in atntr1 mutants revealed a bias for downstream 5′ and 3′ splice site selection and an enhanced rate of exon skipping. A local reduction in PolII occupancy at misspliced exons and introns in atntr1 mutants suggests that directionality in splice site selection is a manifestation of fast PolII elongation kinetics. In agreement with this model, we found AtNTR1 to bind target genes and co-localise with PolII. A minigene analysis further confirmed that strong alternative splice sites constitute an AtNTR1-dependent transcriptional roadblock. Plants deficient in PolII endonucleolytic cleavage showed opposite effects for splice site choice and PolII occupancy compared to atntr1 mutants, and inhibition of PolII elongation or endonucleolytic cleavage in atntr1 mutant resulted in partial reversal of splicing defects. We propose that AtNTR1 is part of a transcription elongation checkpoint at alternative exons in Arabidopsis. Synopsis AtNTR1, the Arabidopsis homologue of spliceosome disassembly factor NTR1, is required for pausing of RNA polymerase II at strong alternative splicing sites. AtNTR1 is required for proper splicing of seed dormancy regulator DOG1. AtNTR1-deficient plants display enhanced RNA PolII elongation rate across strong alternative splice sites. AtNTR1 co-localises with RNA PolII and associates to alternatively spliced target genes. Plants deficient in AtNTR1 and TFIIS show opposite directionality in splice site selection. Introduction Splicing is a highly complicated process that involves more than 200 proteins and five small RNAs associated with the spliceosome at different stages of splicing (Wahl et al, 2009). This enormous number of proteins is reflected in the number of molecular processes, including transcription, that are intertwined with splicing. Alternative splicing is a manifestation of this vast complexity, with more than 95% of human and 60% of Arabidopsis genes showing at least two splicing isoforms (Pan et al, 2008; Filichkin et al, 2009; Marquez et al, 2012). One of the key plant developmental regulators with reported alternative splicing of its pre-mRNA is the DELAY OF GERMINATION 1 protein, DOG1 (Bentsink et al, 2006). The DOG1 expression level is responsible for the delay of germination in freshly harvested seeds and is regulated by various transcription elongation factors (Liu et al, 2007; Grasser et al, 2009), making it a good plant model for studying the crosstalk between splicing and elongation. The interconnection of transcription and splicing has been extensively studied (Howe et al, 2003; Pagani et al, 2003; Chanarat et al, 2011; Close et al, 2012). Several models have been proposed to explain how chromatin regulates alternative splicing, including the direct sensing of histone modifications by spliceosome-associated factors, and influence of the transcription elongation rate on alternative splice site selection. This latter model is known as the kinetic coupling model (De la Mata et al, 2003). It is based on the observation that the changes of RNA polymerase II (PolII) elongation rate affect the selection of alternative splice sites: the slowing down of polymerase leads to exon inclusion and upstream splice site selection, while the acceleration of PolII leads to exon skipping and downstream splice site selection. Recent splicing analysis of a broad list of yeast PolII mutants, with slow and fast elongation kinetics, has confirmed the original model (Braberg et al, 2013). Although the slow PolII leads mainly to exon inclusion, there are several reports where reduced PolII elongation results in increased alternative exon skipping, including exon 9 of CFTR gene (Dutertre et al, 2010; Ip et al, 2011; Dujardin et al, 2014). For this gene, the slow elongation facilitates the binding of a negative regulator to nascent RNA that in turn results in exon skipping rather than exon inclusion (Dujardin et al, 2014). Genetic analyses have suggested that DOG1 is a direct target of TFIIS (Mortensen & Grasser, 2014). TFIIS is an elongation factor (Sekimizu et al, 1976) required for RNA polymerase II processivity both in vitro (Izban & Luse, 1992; Reines, 1992; Cheung & Cramer, 2011) and in vivo (Sigurdsson et al, 2010). In agreement with the kinetic coupling model, tfIIs mutant shows defects in splicing of a reporter gene in yeast (Howe et al, 2003). In addition, transient, splicing-dependent hyper-accumulation of a paused polymerase at the intron was visualised in yeast by the use of synchronised reporter system (Alexander et al, 2010). This transient transcriptional pausing event was suggested to constitute a quality checkpoint imposed by co-transcriptional splicing. This interpretation is consistent with the observed over-accumulation of PolII in humans at alternative introns and exons (Batsché et al, 2005; Saint-André et al, 2011). NTR1 is an accessory spliceosomal component that has been characterised as an interactor of the NineTeen Complex (NTC) in yeast (Tsai, 2005; Agafonov et al, 2011). NTR1 increases PRP43 helicase activity, facilitating intron lariat release. In addition, NTR1 has been proposed to assist in the PRP43-dependent spliceosome quality checkpoint throughout the splicing cycle (Koodathingal et al, 2010; Mayas et al, 2010). In agreement with this, NTR1 has been repeatedly co-purified with the spliceosome at different stages of splicing (Cvitkovic & Jurica, 2013). The spliceosome complex has not been purified in plants, but the Arabidopsis SPLICEOSOMAL TIMEKEEPER LOCUS 1 protein (STIPL1) has been characterised as a homologue of NTR1 (Jones et al, 2012). In agreement with this interpretation, the mutant of the Arabidopsis NTR1 homologue has extensive splicing defects and shows circadian clock defects due to the missplicing of one of the circadian clock genes (Jones et al, 2012). Surprisingly, purification of the human NTR1 complex has revealed that, in addition to its interaction with PRP43, it is co-purified with conserved group of proteins containing GCFC domain (GC-rich sequence DNA-binding factor): C2ORF3 and GCFC (Yoshimoto et al, 2014). The closest Arabidopsis homologue of C2ORF3 and GCFC is ILP1, a protein shown to bind DNA and to control endoreduplication (Yoshizumi et al, 2006). Here, we report that the Arabidopsis thaliana NTR1 homologue (AtNTR1) is crucial for DOG1 expression and splicing. Analysis of splicing defects of DOG1 and other genes shows a strong bias towards downstream splice site selection in atntr1. In accordance with kinetic coupling model, we hypothesise that this bias is a consequence of fast PolII elongation at the splice sites in atntr1 mutant. Our PolII ChIP data revealed localised decrease in PolII occupancy at affected splice sites. This result is interpreted by us as a localised change in elongation rate. We were unable to reproduce this phenomenon using neither chemical modulation of splicing, nor mutants in other splicing factors, which proves that localised decrease in PolII occupancy is AtNTR1-specific. This interpretation is consistent with observed immuno-co-localisation of AtNTR1 with PolII in the nucleus and the presence of AtNTR1 at DNA of its target genes as shown by ChIP. Analysis of AtNTR1-dependent splicing events showed that NTR1 is required for splicing of strong, consensus-like, alternative splice sites. This was corroborated by mutational analysis that showed an atntr1-dependent increased accumulation of PolII ChIP signal at the strong alternative splice sites. Our data are consistent with NTR1 being required for co-transcriptional pausing of polymerase at strong alternative splice sites. We therefore interpret the directionality of alternative splicing defects in atntr1 mutant as a manifestation of PolII elongation defects. The role of transcription elongation in alternative splice site selection has been extensively studied (De la Mata et al, 2011). To investigate whether alternative splice site selection in plants also depends on transcription elongation rate, we have compromised PolII elongation by mutating TFIIS and exposed plants to 6AU (6-azauracil) and MPA (mycophenolic acid) treatment. Observed changes in alternative splicing were predominantly opposite to ones observed in atntr1 mutant and consistent with prediction based on the kinetic coupling model, supporting our conclusions. Results AtNTR1 regulates seed dormancy, DOG1 expression and splicing The NTR1 homologue in Arabidopsis was originally identified by means of genetic screen aimed to identify circadian clock regulators (Jones et al, 2012). We found that in addition to its role in circadian clock regulation, atntr1-1 mutants showed pleiotropic phenotypes including low seed dormancy, altered flowering time, altered leaf morphology and enhanced lethality at elevated temperatures (Fig 1A, Supplementary Fig S1). We focused on the seed dormancy phenotype and confirmed that both the available alleles, atntr1-1 and atntr1-2, showed enhanced germination without stratification (Fig 1A and B, Supplementary Fig S1A). Interestingly, dog1 mutants have been shown to have similar phenotype (Bentsink et al, 2006). In agreement with the low seed dormancy phenotype, we found reduced expression of DOG1 gene in atntr1 mutants, both in seeds (Fig 1C) and seedlings (Supplementary Fig S1B). Because AtNTR1 deficiency leads to massive splicing defects in Arabidopsis (Jones et al, 2012) and DOG1 is a subject of alternative splicing (Bentsink et al, 2006), we analysed the splicing defects of the DOG1 transcripts. The atntr1-1 mutation resulted in more pronounced usage of the 5′ downstream splice site, with a concomitant reduction in the upstream 5′SS selection in comparison with wild-type plants (Fig 1D). Additionally, an approximately 50% increase in intron retention was observed (Fig 1D). The altered splicing isoforms corresponded to the most abundant splice isoforms of DOG1, namely alpha and beta (Bentsink et al, 2006; Schwab, 2008). Consequently, we have measured all four isoforms reported for DOG1 and found that, indeed, isoforms alpha and beta were the most affected (Supplementary Fig S1C and D). Figure 1. AtNTR1 mutation results in reduced seed dormancy, low DOG1 expression and a tendency towards downstream splice site selection A, B. Photographs (A) and quantification (B) of seed dormancy tests. The chart represents the average percentage of germinated seeds without stratification after 4 days of growth in LD. The error bars represent ± SE (n = 3). Tests were performed on freshly harvested seeds, with or without 3 days of stratification growth in LD. C. qPCR of DOG1 expression in siliques (16 days after pollination). The graph represents the average ratio of DOG1 to UBC, normalised to Col-0 (WT). The error bars represent ± SE (n = 3). D. DOG1 splicing was assessed by RT–PCR combined with capillary electrophoresis. The graph represents the mean relative contribution of the mRNA forms found in the total pool of amplified products. The black and grey bars represent the data for Col-0 and atntr1-1, respectively. The error bars represent ± SD (n = 3). To the right of the charts, the structures of the examined transcripts are shown (black boxes, constitutive exons; white boxes, alternative regions; black lines, introns). The black arrows show the locations of primers. Downstr. and upstr. stand for downstream and upstream splicing event, respectively. E. Directionality of splice site selection in atntr1. Splicing was analysed in 14-day-old MS grown plants. For each type of alternative splice event, the black and white boxes show the contributions of opposite direction splicing events. The numbers represent the percentage of splice events supporting the direction of the splice site event change (also shown on horizontal axis). The numbers on the right-hand panel represent the number of affected splicing events versus total number of splicing events analysed. The white bars represent distal 3′ and downstream 5′ splice site selection (3′SS/5′SS), exon skipping (ES) and intron retention (IR), while the black bars represent 5′ and 3′ splice site selection (3′SS/5′SS), exon inclusion (ES) and intron splicing (IR). Download figure Download PowerPoint The atntr1 mutant shows bias in alternative 5′ and 3′ splice site selection We were intrigued by the change in splice site selection on DOG1 towards the downstream splice site. DOG1 expression strongly depends on factors required for efficient transcription elongation (including TFIIS). The observed tendency towards downstream splice site selection in the NTR1 mutant could be a manifestation of a defect in the PolII elongation rate (Liu et al, 2007; Mortensen & Grasser, 2014). We therefore extended our observation of alternative splicing changes. Independently of the previous report, a selection of 144 alternative splice events were analysed in atntr1-1 and in wild-type plants (Jones et al, 2012). In agreement with previous results, we found that the most abundant splicing defects were intron retention and exon skipping (Fig 1E). For 144 alternative splicing events analysed, 74 were significantly changed. Prominent bias in the directionality of alternative 5′ splice site selection was observed, which is in accordance with the directionality of splice site selection on DOG1 (Fig 1D and E). Of 16 affected alternative 3′ splice site selection events, 14 (88%) were changed in atntr1-1 towards downstream splice sites (SS). Similar bias was also observed in the directionality of splicing events in the case of 5′ splice site events (seven of 10 changed towards downstream SS—70%) and in exon skipping events (18 of 20 changed towards exon skipping—90%) (Fig 1E and Supplementary Table S1). Upstream/downstream splice site selection has been proposed to represent a manifestation of the polymerase II elongation rate (De la Mata et al, 2003, 2011). Consequently, the observed bias could indicate a defect in transcription elongation across the affected splice sites in the atntr1-1 mutant. AtNTR1 is required for splicing of strong consensus splice sites Next, we wanted to understand what creates specificity for AtNTR1 at some splice sites but not the others. Analysis of acceptor splice sites sequences showed no clear difference between AtNTR1-dependent and AtNTR1-independent introns (Supplementary Fig S2B). On the other hand, analysis of donor sites revealed a significant difference at position +3/+4 (P-value < 0.05, Fisher's exact test). AtNTR1-dependent introns show a higher likelihood of A/G at +3 and A at +4 positions compared to AtNTR1-independent splice sites (Supplementary Fig S2A). The consensus donor splicing site sequence in Arabidopsis is AG|GTAAGT. Consequently, the affected introns more closely resemble the whole-genome consensus than introns with splicing unaffected in atntr1-1. We decided to test whether the AtNTR1 requirement for splicing is specified by the strong sequence of alternative splicing donor splice site as has been suggested by our sequence analysis. We selected an alternative 5′SS event that is not dependent on NTR1 and has week consensus sequences at the upstream and downstream splicing sites. Subsequently, those sites were mutated into strong consensus sequences by changing two nucleotides at each site (Supplementary Fig S1C). Analysis showed that whereas splicing of the native version (5′SS wt) was not changed in atntr1 mutant, the splicing of the mutated construct (5′SS strong) was affected (Supplementary Fig S1D). Although the change in alternative splice site selection was modest, it was statistically significant (t-test P-value < 0.01). This confirms our initial observation that the 5′SS consensus with extended homology to U1 constitutes a preferable target for NTR1 splicing activity. In addition, this analysis shows that the AtNTR1 effect on splicing is downstream of splice site recognition by the spliceosome, which is consistent with the AtNTR1 role in recycling of U6. In addition to U6/5/2 snRNPs, AtNTR1 interacts with U1 To obtain more insights into the potential function of AtNTR1, RNA molecules associated with AtNTR1 were analysed using RNA immunoprecipitation (RIP) followed by RT–PCR. In this experiment, a complementing transgenic Arabidopsis line expressing the AtNTR1-GFP fusion protein in the atntr1-1 genetic background was used with antibodies recognising GFP. Given the well-documented role of NTR1 in U6, U5 and U4 recycling, we tested AtNTR1 interaction with those molecules. We could clearly observe an enrichment of U6, U5 and U2 RNA in the AtNTR1-GFP-immunoprecipitated fraction, compared to our negative control (Fig 2A). Surprisingly, a strong and reproducible interaction of AtNTR1 with U1 was also observed (Fig 2A). In contrast, U3 and 18S rRNA showed no enrichment, confirming the stringency of our method (Fig 2A). This result was further confirmed by the identification of AtNTR1-associated proteins by means of mass spectrometry. The U1 associated protein—U1A—was one of our highest-scoring interactors (Table 1). Table 1. AtNTR1 co-purifying proteins. Gene ID Gene name MW (Da) Number of unique peptides P1-P2-P3-P4 AT1G17070 AtNTR1 96,937 37-36-26-30 AT5G08550 ILP1 100,998 21-22-30-28 AT1G24180 IAR4 43,787 1-2-2-2 AT2G39770 CYT1 39,837 1-1-1-2 AT2G47580 U1A 58,456 1-1-1-1 AT2G30050 WD40 32,907 1-1-1-1 Seedlings of complementing AtNTR1-GFP transgenic lines, expressed under native promoter, were used for four independent purifications with three negative controls (Col-0). After trypsin digestion and mass spectrometry, proteins identified in all purifications but not in negative controls were listed in the table. Number of unique peptides matching each identified protein is shown separately for each purification (P1–4). Figure 2. AtNTR1 interacts with U6 and U1 snRNA and co-localise with PolII Electrophoresis of RT–PCR products showing interactions of AtNTR1 with selected snRNA targets detected by RIP. The level of transcripts co-precipitated from transgenic plants expressing ANTR1-GFP (IP+) or wild-type plants (IP−) using anti-GFP antibody was measured by RT–PCR normalised to the inputs. To control the amplification from gDNA, controls without reverse transcriptase (RT) were performed. U3 snRNA and 18S rRNA were used as negative controls for interaction. Fluorescent immunostaining of nuclei showing the co-localisation of AtNTR1 with SC35, total PolII, Ser5-phosphorylated PolII (PolIIA) or Ser2-phosphorylated PolII (PolIIO). AtNTR1 was detected using an antibody raised against AtNTR1 peptide. Scale bar represents 2.5 μm. Download figure Download PowerPoint ILP1, a GCFC domain-containing protein, interacts with AtNTR1 and is required for efficient splicing The highest-ranking NTR1 interactor on our list was a protein known as ILP1 in Arabidopsis (Yoshizumi et al, 2006). ILP1 contains the GCFC domain (GC-rich sequence DNA-binding factor-like domain) and is a homologue of the human proteins C2ORF3 and GCFC (also known as Pax3/7BP) (Diao et al, 2012; Yoshimoto et al, 2014). Both, the bimolecular fluorescence complementation (BiFC) assay using YFP (Supplementary Fig S3B) and an yeast two-hybrid assay (Supplementary Fig S3C), confirmed our original finding and suggested direct AtNTR1–ILP1 interaction. Interestingly, homologues of ILP1 in human co-purify with TFIP11, a human homologue of AtNTR1 (Yoshimoto et al, 2014). This indicates that this interaction is conserved between species, which suggests that it may be important for NTR1 function. ILP1 in Arabidopsis binds to a promoter of a key cell cycle gene and controls its expression, providing a possible explanation for endoreduplication defects in the ilp1 mutant (Yoshizumi et al, 2006). In addition, human homologues of ILP1 likewise bind to gene promoters to regulate their expression (Diao et al, 2012). Next, we tested whether ILP1 was involved in splicing regulation in Arabidopsis. The atntr1-1 and ilp1-1 mutants' analysis revealed that ilp1-1 had very strong splicing defects, with virtually all splicing events affected in atntr1-1 also being misregulated in the ilp1 mutant (Supplementary Fig S3D, Supplementary Table S2). This finding is consistent with the recent data on a human ILP1 homologue showing that the depletion of C2ORF3 by RNAi repressed pre-mRNA splicing in vitro (Yoshimoto et al, 2014). We conclude that ILP1, like its human homologues, is a direct interactor of AtNTR1 and that GCFC domain-containing proteins are required for efficient splicing both in Arabidopsis and in humans. PolII co-localises with AtNTR1 AtNTR1 immunolocalisation was investigated, to test the relationship between AtNTR1 and PolII. We confirmed previous results from human cells showing that NTR1 is localised in the nucleus but excluded from the nucleolus, using a complementing genomic NTR1-GFP line (Supplementary Fig S1I and J) and AtNTR1 antibody (Fig 2B) (Tannukit et al, 2008). In addition, it was found that AtNTR1 is only partially co-localised with the SC35 splicing factor using dual labelling (Fig 2B), which is in agreement with results concerning NTR1 mouse homologue (Wen et al, 2005). Subsequently, we investigated the co-localisation of AtNTR1 with PolII. Three different PolII antibodies were used: the first recognising all forms of PolII (total PolII), the second recognising the Ser5-phosphorylated form of PolII (PolIIA) that is usually associated with the initiation of transcription, and the third recognising Ser2-phosphorylated PolII (PolIIO), which is believed to primarily mark PolII associated with gene bodies. Partial co-localisation with total PolII was observed. It could be attributed to the Ser2-phosphorylated form of PolII, based on observed no co-localisation with PolIIA and strong co-localisation of AtNTR1 with the PolIIO (Fig 2B). Although the functional distinction between Ser5-phosphorylated and Ser2-phosphorylated PolII is not absolute, it is generally believed that Ser2 phosphorylation is a mark of elongating polymerase (Komarnitsky, 2000; Buratowski, 2009). AtNTR1 acts co-transcriptionally at affected splice sites The co-localisation of AtNTR1 with Ser2-phosphorylated PolII suggests that NTR1 can be physically present at the target genes. In order to test this, AtNTR1 localisation on the DOG1 gene was analysed by chromatin immunoprecipitation (ChIP) using antibodies that recognise AtNTR1. Our ChIP analysis shows that AtNTR1 is present at the gene body and promoter of DOG1, in contrast to an intergenic region selected as a negative control (Fig 3). In addition, a set of five other genes was tested for NTR1 presence. Clear NTR1 signal could be detected on all of them (Supplementary Fig S4). The genes were selected from set that displayed misregulated alternative splicing in atntr1. Moreover, the five genes were chosen to represent different types of alternative splicing events. The physical presence of AtNTR1 on those genes substantiates our NTR1 PolII co-localisation data and suggests that at least some of NTR1 activity is happening co-transcriptionally. Figure 3. AtNTR1 is present at the DOG1 geneAtNTR1 antibodies were used to analyse AtNTR1 protein presence at DOG1 locus using ChIP. Data shown represent enrichment above background level measured in atntr1-1 mutant. Gene structure is shown with black boxes representing constitutive exons; grey box, alternative region; white box, promoter region; black lines, introns. Red lines show amplified regions. 0.5 kb scale is shown. Error bars represent ± SD of three independent experiments. As an additional negative control, primers amplifying an unlinked intergenic region (IGR) were used. Download figure Download PowerPoint We conclude that AtNTR1 is present at or close to DNA of genes that display AtNTR1-dependent splicing. In addition, our ChIP analysis shows that AtNTR1 is present throughout the analysed genes, with no clear enrichment at misspliced introns. This result is consistent with immuno-co-localisation studies of AtNTR1 and PolII. Localised PolII level reduction in atntr1 mutant Our data show a strong link between AtNTR1 and PolII. Moreover, the splicing defects observed in the NTR1 mutant could be a manifestation of fast PolII elongation across these splice sites. We therefore considered a possibility that AtNTR1 is involved in the control of PolII at those splice sites. To address this, the genes that previously showed to be direct AtNTR1 targets were used to assess PolII profile by ChIP using antibodies that recognise total PolII. Analysis of PolII occupancy in atntr1-1 consistently revealed a significant reduction in PolII for all genes with AtNTR1-dependent splicing (five of the five genes tested) (Fig 4 and Supplementary Fig S5). With an exception of DOG1, the reduction was limited to or strongest at the regions of misspliced introns. The At5g04430 gene was analysed as a control, as it shows atntr1-independent alternative splicing and consequently showed no significant reduction in PolII levels in atntr1 mutant. Figure 4. atntr1, in contrast to herboxidiene-treated plants, shows localised decrease in PolII occupancy o
Referência(s)