Abstract
The serine and arginine-rich protein family (SR proteins) are highly conserved regulators of pre-mRNA splicing. SF2/ASF, a prototype member of the SR protein family, is a multifunctional RNA binding protein with roles in pre-mRNA splicing, mRNA export and mRNA translation. These observations suggest the intriguing hypothesis that SF2/ASF may couple splicing and translation of specific mRNA targets in vivo. Unfortunately the paucity of endogenous mRNA targets for SF2/ASF has hindered testing of this hypothesis. Here, we identify endogenous mRNAs directly cross-linked to SF2/ASF in different sub-cellular compartments. Cross-Linking Immunoprecipitation (CLIP) captures the in situ specificity of protein-RNA interaction and allows for the simultaneous identification of endogenous RNA targets as well as the locations of binding sites within the RNA transcript. Using the CLIP method we identified 326 binding sites for SF2/ASF in RNA transcripts from 180 protein coding genes. A purine-rich consensus motif was identified in binding sites located within exon sequences but not introns. Furthermore, 72 binding sites were occupied by SF2/ASF in different sub-cellular fractions suggesting that these binding sites may influence the splicing or translational control of endogenous mRNA targets. We demonstrate that ectopic expression of SF2/ASF regulates the splicing and polysome association of transcripts derived from the SFRS1, PABC1, NETO2 and ENSA genes. Taken together the data presented here indicate that SF2/ASF has the capacity to co-regulate the nuclear and cytoplasmic processing of specific mRNAs and provide further evidence that the nuclear history of an mRNA may influence its cytoplasmic fate.
Highlights
Eukaryotic messenger RNA must be processed prior to programming protein synthesis
Cross-linking immunoprecipitation of SF2/ASF Previously, we have shown that SF2/ASF binds directly to cytoplasmic messenger RNA (mRNA) in vivo and enhances the translation of reporter mRNAs both in vitro and in vivo [8], suggesting that SF2/ASF may regulate the nuclear and cytoplasmic fate of specific endogenous mRNAs
In order to determine if SF2/ASF can regulate nuclear and cytoplasmic processing of endogenous mRNAs we used the Cross-Linking Immunoprecipitation (CLIP) protocol to identify binding sites for SF2/ASF in mRNAs from different subcellular fractions
Summary
Eukaryotic messenger RNA (mRNA) must be processed prior to programming protein synthesis. The minimal modifications for most mRNAs include capping, pre-mRNA splicing and polyadenylation [1]. These reactions occur in the nucleus and must be completed prior to nuclear export of the mRNA to the cytoplasm. RNA processing reactions have been extensively studied using biochemical systems; these are functionally linked in living cells providing increased efficiency and regulatory potential for gene expression [3]. The molecular mechanisms responsible for the coupling of post-transcriptional regulatory networks are poorly understood. A subset of multi-functional mRNA binding proteins operating at the interface of distinct RNA processing machineries may contribute to coupling of post-transcriptional gene expression
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.