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The spliceosome deposits multiple proteins 20-24 nucleotides upstream of mRNA exon-exon junctions
H Le Hir1, E Izaurralde, L E Maquat
1Howard Hughes Medical Institute, Department of Biochemistry, Brandeis University, 415 South Street, Waltham, MA 02454, USA.
Abstract:
Eukaryotic mRNAs exist in vivo as ribonucleoprotein particles (mRNPs). The protein components of mRNPs have important functions in mRNA metabolism, including effects on subcellular localization, translational efficiency and mRNA half-life. There is accumulating evidence that pre-mRNA splicing can alter mRNP structure and thereby affect downstream mRNA metabolism. Here, we report that the spliceosome stably deposits several proteins on mRNAs, probably as a single complex of approximately 335 kDa. This complex protects 8 nucleotides of mRNA from complete RNase digestion at a conserved position 20-24 nucleotides upstream of exon-exon junctions. Splicing-dependent RNase protection of this region was observed in both HeLa cell nuclear extracts and Xenopus laevis oocyte nuclei. Immunoprecipitations revealed that five components of the complex are the splicing-associated factors SRm160, DEK and RNPS1, the mRNA-associated shuttling protein Y14 and the mRNA export factor REF. Possible functions for this complex in nucleocytoplasmic transport of spliced mRNA, as well as the nonsense-mediated mRNA decay pathway, are discussed.
Insights
The spliceosome deposits a protein complex onto eukaryotic mRNAs during splicing. This complex protects specific mRNA regions, influencing mRNA metabolism and transport.
Area of Science:
- Molecular Biology
- RNA Biology
- Gene Expression
Background:
- Eukaryotic messenger RNAs (mRNAs) function as ribonucleoprotein particles (mRNPs) in vivo.
- mRNP protein components are crucial for mRNA metabolism, affecting localization, translation, and stability.
- Evidence suggests pre-mRNA splicing influences mRNP structure and subsequent mRNA metabolism.
Purpose of the Study:
- To investigate the direct deposition of proteins onto mRNA by the spliceosome.
- To characterize the composition and properties of the spliceosome-deposited complex.
- To explore the functional implications of this complex in mRNA metabolism.
Main Methods:
- RNase digestion assays in HeLa cell nuclear extracts and Xenopus laevis oocyte nuclei.
- Immunoprecipitation to identify protein components of the deposited complex.
- Analysis of protected mRNA regions relative to exon-exon junctions.
Main Results:
- The spliceosome stably deposits a protein complex (approx. 335 kDa) onto spliced mRNAs.
- This complex confers RNase protection to a specific 8-nucleotide region (20-24 nucleotides upstream of exon-exon junctions).
- Five identified components include SRm160, DEK, RNPS1, Y14, and REF.
Conclusions:
- The spliceosome deposits a stable protein complex on spliced mRNAs, impacting mRNA processing.
- This complex may play roles in nucleocytoplasmic transport of mature mRNA.
- Potential involvement in the nonsense-mediated mRNA decay (NMD) pathway is suggested.