NTR 1 is required for transcription elongation checkpoints at alternative exons in Arabidopsis

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 0 and 3 0 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.

The Composition of the Arabidopsis RNA Polymerase II Transcript Elongation Complex Reveals the Interplay between Elongation and mRNA Processing Factors

“…For example, the RNAPII elongation rate, which is under control of TEFs, influences the efficiency of splicing and polyadenylation events (Elkon et al, 2013;Saldi et al, 2016). In plants, the interplay between transcript elongation and mRNA processing has only recently become apparent, as exemplified by the connection of TFIIS and PAF1-C with splicing (Dolata et al, 2015;Li et al, 2016). We also observed that a range of splicing factors copurified with CstF77.…”

Salt Stress Reveals a New Role for ARGONAUTE1 in miRNA Biogenesis at the Transcriptional and Posttranscriptional Levels

“…ChIP in the Arabidopsis AGO1:FLAG line using anti-FLAG antibodies. Data normalized to a CTD Ser-5 is an indication of enzyme pausing during transcription (Alexander et al, 2010;Dolata et al, 2015). Therefore, we performed the stress experiments in the cpl1-7 mutant.…”

“…ChIP was performed as previously described in Dolata et al (2015) using 2 mg of each antibody per IP: anti-RNAPII (AS11 1804; Agrisera), anti-AGO1 (AS09 527; Agrisera), and 30 mL of ANTI-FLAG M2 Magnetic Beads (SigmaAldrich). The RNase+ nuclear extract sample was treated with 2.5 mg of RNase A (Thermo Fisher Scientific) during IP.…”

Control of seed dormancy in Arabidopsis by a cis -acting noncoding antisense transcript

“…Transcription factor (TF)IIS knockout and TFIIS dominant-negative mutation lead to the slowdown of PolII elongation, which enhances proximal splice site selection on DOG1. In contrast, mutation of the spliceosome cofactor AtNTR1, which increases the rate of PolII elongation, results in the selection of distal splice sites (14). In addition to changes in DOG1 splice site selection, tfIIs and atntr1 mutants display low DOG1 expression and consequently a weak seed dormancy phenotype (14)(15)(16).…”

“…In contrast, mutation of the spliceosome cofactor AtNTR1, which increases the rate of PolII elongation, results in the selection of distal splice sites (14). In addition to changes in DOG1 splice site selection, tfIIs and atntr1 mutants display low DOG1 expression and consequently a weak seed dormancy phenotype (14)(15)(16). Other known factors required for high DOG1 expression include the histone H2B ubiquitin transferases HUB1 and HUB2 (17),…”