Signalling and Response Daily temperature cycles promote alternative splicing of RNAs encoding SR45a, a splicing regulator in maize

Elevated temperatures enhance alternative RNA splicing in maize (Zea mays) with the potential to expand the repertoire of plant responses to heat stress. Alternative RNA splicing generates multiple RNA isoforms for many maize genes, and here we observed changes in the pattern of RNA isoforms with temperature changes. Increases in maximum daily temperature elevated the frequency of the major modes of alternative splices (AS), in particular retained introns and skipped exons. The genes most frequently targeted by increased AS with temperature encode factors involved in RNA processing and plant development. Genes encoding regulators of alternative RNA splicing were themselves among the principal AS targets in maize. Under controlled environmental conditions, daily changes in temperature comparable to field conditions altered the abundance of different RNA isoforms, including the RNAs encoding the splicing regulator SR45a, a member of the SR45 gene family. We established an “in protoplast” RNA splicing assay to show that during the afternoon on simulated hot summer days, SR45a RNA isoforms were produced with the potential to encode proteins efficient in splicing model substrates. With the RNA splicing assay, we also defined the exonic splicing enhancers that the splicing-efficient SR45a forms utilize to aid in the splicing of model substrates. Hence, with rising temperatures on hot summer days, SR45a RNA isoforms in maize are produced with the capability to encode proteins with greater RNA splicing potential.

Splicing Characteristics of Dystrophin Pseudoexons and Identification of a Novel Pathogenic Intronic Variant in the DMD Gene

Pseudoexon (PE) inclusion has been implicated in various dystrophinopathies; however, its splicing characteristics have not been fully investigated. This study aims to analyze the splicing characteristics of dystrophin PEs and compare them with those of dystrophin canonical exons (CEs). Forty-two reported dystrophin PEs were divided into a splice site (ss) group and a splicing regulatory element (SRE) group. Five dystrophin PEs with characteristics of poison exons were identified and categorized as the possible poison exon group. The comparative analysis of each essential splicing signal among different groups of dystrophin PEs and dystrophin CEs revealed that the possible poison exon group had a stronger 3′ ss compared to any other group. As for auxiliary SREs, different groups of dystrophin PEs were found to have a smaller density of diverse types of exonic splicing enhancers and a higher density of several types of exonic splicing silencers compared to dystrophin CEs. In addition, the possible poison exon group had a smaller density of 3′ ss intronic splicing silencers compared to dystrophin CEs. To our knowledge, our findings indicate for the first time that poison exons might exist in DMD (the dystrophin gene) and present with different splicing characteristics than other dystrophin PEs and CEs.

SRSF2 regulation of MDM2 reveals splicing as a therapeutic vulnerability of the p53 pathway

ABSTRACTMDM2 is an oncogene and critical negative regulator of tumor suppressor p53. Genotoxic stress causes alternative splicing of MDM2 transcripts, which leads to alterations in p53 activity and contributes to tumorigenesis. MDM2-ALT1 is one of transcripts predominantly produced in response to genotoxic stress and is comprised of terminal coding exons 3 and 12. Previously, we found that SRSF1 induces MDM2-ALT1 by promoting MDM2 exon 11 skipping. Here we report that splicing regulator SRSF2 antagonizes the regulation of SRSF1 by facilitating the inclusion of exon 11 through binding at two conserved exonic splicing enhancers. Overexpression of SRSF2 reduced the generation of MDM2-ALT1 in genotoxic stress condition, whereas knockdown induces the expression of MDM2-ALT1 in absence of genotoxic stress. Consistently, blocking the exon 11 SRSF2 binding sites using oligonucleotides promotes MDM2-ALT1. The regulation of MDM2 splicing by SRSF2 is also conserved in mouse as mutation of one SRSF2 binding site in Mdm2 exon 11, using CRISPR-Cas9, increases the expression MDM2-ALT1 homolog Mdm2-MS2 and proliferation of NIH 3T3 cells. Taken together, these findings underscore the relevance of MDM2 alternative splicing in cancer and suggest that p53 levels can be modulated by artificially regulating MDM2 splicing.

Intronless mRNAs transit through nuclear speckles to gain export competence

Nuclear speckles (NSs) serve as splicing factor storage sites. In this study, we unexpectedly found that many endogenous intronless mRNAs, which do not undergo splicing, associate with NSs. These associations do not require transcription, polyadenylation, or the polyA tail. Rather, exonic splicing enhancers present in intronless mRNAs and their binding partners, SR proteins, promote intronless mRNA localization to NSs. Significantly, speckle targeting of mRNAs promotes the recruitment of the TREX export complex and their TREX-dependent nuclear export. Furthermore, TREX, which accumulates in NSs, is required for releasing intronless mRNAs from NSs, whereas NXF1, which is mainly detected at nuclear pores, is not. Upon NXF1 depletion, the TREX protein UAP56 loses speckle concentration but coaccumulates with intronless mRNAs and polyA RNAs in the nucleoplasm, and these RNAs are trapped in NSs upon UAP56 codepletion. We propose that the export-competent messenger RNP assembly mainly occurs in NSs for intronless mRNAs and that entering NSs serves as a quality control step in mRNA export.

Intron gain by tandem genomic duplication: a novel case and a new version of the model

Origin and subsequent accumulation of spliceosomal introns are prominent events in the evolution of eukaryotic gene structure. Recently gained introns would be especially useful for the study of the mechanisms of intron gain because randomly accumulated mutations might erase the evolutionary traces. The mechanisms of intron gain remain unclear due to the presence of very few solid cases. A widely cited model of intron gain is tandem genomic duplication, in which the duplication of an AGGT-containing exonic segment provides the GT and AG splicing sites for the new intron. We found that the second intron of the potato RNA-dependent RNA polymerase gene PGSC0003DMG402000361 originated mainly from a direct duplication of the 3′ side of the upstream intron. The 5' splicing site of this new intron was recruited from the upstream exonic sequence. In addition to the new intron, a downstream exonic segment of 178 bp also arose from duplication. Most of the splicing signals were inherited directly from the parental intron/exon structure, including a putative branch site, the polypyrimidine tract, the 3′ splicing site, two putative exonic splicing enhancers and the GC contents differentiated between the intron and exon. We propose a new version of the tandem genomic duplication model, termed as the partial duplication of the preexisting intron/exon structure. This new version and the widely cited version are not mutually exclusive.

Intron gain by tandem genomic duplication: a novel case and a new version of the model

Origin and subsequent accumulation of spliceosomal introns are prominent events in the evolution of eukaryotic gene structure. Recently gained introns would be especially useful for the study of the mechanisms of intron gain because randomly accumulated mutations might erase the evolutionary traces. The mechanisms of intron gain remain unclear due to the presence of very few solid cases. A widely cited model of intron gain is tandem genomic duplication, in which the duplication of an AGGT-containing exonic segment provides the GT and AG splicing sites for the new intron. We found that the second intron of the potato RNA-dependent RNA polymerase gene PGSC0003DMG402000361 originated mainly from a direct duplication of the 3′ side of the upstream intron. The 5' splicing site of this new intron was recruited from the upstream exonic sequence. In addition to the new intron, a downstream exonic segment of 178 bp also arose from duplication. Most of the splicing signals were inherited directly from the parental intron/exon structure, including a putative branch site, the polypyrimidine tract, the 3′ splicing site, two putative exonic splicing enhancers and the GC contents differentiated between the intron and exon. We propose a new version of the tandem genomic duplication model, termed as the partial duplication of the preexisting intron/exon structure. This new version and the widely cited version are not mutually exclusive.

Intron gain by tandem genomic duplication: a novel case and a modification of the traditional model

Origin and subsequent accumulation of spliceosomal introns are prominent events in the evolution of eukaryotic gene structure. Recently gained introns would be especially useful for the study of the mechanism(s) of intron gain because the evolutionary traces might have not been erased by randomly accumulated mutations. However, the mechanism(s) of intron gain remain unclear due to the presence of a few solid cases. A widely cited model of intron gain is tandem genomic duplication, in which the duplication of an AGGT-containing exonic segment provides the GT and AG splicing sites for the new intron. However, successful recognition and splicing of an intron require many more signals than those at the two splicing sites. We found that the second intron of the potato RNA-dependent RNA polymerase gene PGSC0003DMG402000361 is absent in the orthologous genes of other Solanaceae plants, and sequence similarity showed that the major part of the new intron is a direct duplication of the 3' side of the upstream intron. In addition to the new intron, a downstream exonic segment of 168bp has also been duplicated. Most of the splicing signals were inherited from the parental intron/exon structure, including a putative branch site, the polypyrimidine tract, the 3' splicing site, two putative exonic splicing enhancers and the GC contents differentiated between the intron and exon. We propose a modified version of the tandem genomic duplication model, termed as the partial duplication of the preexisting intron/exon structure.