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A method for the factor-specific spatio-temporal control of splicing

ABSTRACT: Precursor messenger RNA splicing is a highly regulated process, mediated by a complex RNA-protein machinery, the spliceosome, that encompasses several hundred proteins and five small nuclear RNAs in human. Emerging evidence suggests that the spatial organization of splicing factors and their spatio-temporal dynamics participate in the regulation of splicing. For instance, Cajal bodies and nuclear speckles function as centers for snRNP assembly and recycling or storage, respectively, whereas the assembly of spliceosomes on nascent pre-mRNAs predominantly occurs in peri-chromatin fibrils. So far, methods to manipulate the spatial distribution of splicing factors in a temporally defined manner in living cells are missing. Here, we describe such an approach that takes advantage of a reversible chemical dimerizer, and outline the requirements for efficient, reversible re-localization of splicing factors to selected sub-nuclear compartments. In a proof-of-principle study, the partial re-localization of the PRPF38A protein to the nuclear lamina induced a moderate increase in intron retention. Our approach allows fast and reversible re-localization of splicing factors, has few side effects and can be applied to any splicing factor by fusion of a protein tag through genome engineering. Apart from the systematic analysis of the spatio-temporal aspects of splicing regulation, the approach has a large potential for the fast induction and reversal of splicing switches and can reveal mechanisms of splicing regulation in native nuclear environments.

ORGANISM(S): Homo sapiens

PROVIDER: GSE182412 | GEO | 2022/06/18

REPOSITORIES: GEO

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Project description:We applied the RNA-Seq approach to reconstruct the transcriptome of Vitis vinifera cv. Corvina, using RNA pooled from a comprehensive set of sampled tissues in different organs and development steps, and we were able to reconstruct some novel and putative private Corvina genes. We analyzed the expression of these genes in three berry developmental conditions, and posit that they may play some role in the formation of the mature organ. Background: Plants display a high genetic and phenotypic variability among different cultivars. Understanding the genetic components that contribute to phenotypic diversity is necessary to disentangle genetic factors from the environment. Given the high degree of genetic diversity among plant cultivars a whole-genome sequencing and re-annotation of each variety is required but a reliable genome assembly is hindered by the high heterozigosity and sequence divergence. Results: we show the feasibility of an approach based on sequencing of cDNA by RNA-Seq to analyze varietal diversity between a local grape cultivar Corvina and the PN40024 grape reference genome. We detected 15,260 known genes and we annotated alternative splicing isoforms for 9,463 genes. Our approach allowed to define 2,321 protein coding putative novel genes in unannotated or unassembled regions of the reference genome PN40024 and 180 putative private Corvina genes whose sequence is not shared with the reference genome. Conclusions: With a de novo assembly based approach we were able to reconstruct a substantial part of the Corvina transcriptome and we improved substantially known genes annotations by better defining the structure of known genes, annotating splicing isoforms and detecting unannotated genes. Moreover our results clearly define sets of private genes which are likely part of the âdispensableâ genome and potentially involved into influencing some cultivar-specific characteristics. In plant biology a transcriptome de novo assembly approach should not be limited to species where no reference genome is available as it can improve the annotation lead to the identification of genes peculiar of a cultivar.

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A method for the factor-specific spatio-temporal control of splicing

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