Keil, Philipp, Wulf, Alexander, Kachariya, Nitin, Reuscher, Samira, Huhn, Kristin, Silbern, Ivan, Altmuller, Janine ORCID: 0000-0003-4372-1521, Keller, Mario, Stehle, Ralf, Zarnack, Kathi ORCID: 0000-0003-3527-3378, Sattler, Michael ORCID: 0000-0002-1594-0527, Urlaub, Henning and Straesser, Katja (2023). Npl3 functions in mRNP assembly by recruitment of mRNP components to the transcription site and their transfer onto the mRNA. Nucleic Acids Res., 51 (2). S. 831 - 852. OXFORD: OXFORD UNIV PRESS. ISSN 1362-4962

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Abstract

RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large ribonucleoprotein particles (RNPs) these proteins function in. Using UV-induced RNA-protein crosslinking of entire cells, protein complex purification and mass spectrometric analysis, we identified >100 in vivo RNA crosslinks in 16 nuclear mRNP components in Saccharomyces cerevisiae. For functional analysis, we chose Npl3, which displayed crosslinks in its two RNA recognition motifs (RRMs) and in the connecting flexible linker region. Both RRM domains and the linker uniquely contribute to RNA recognition as revealed by NMR and structural analyses. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains. Notably, an npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of other mRNP components into nuclear mRNPs, establishing a so far unknown function of Npl3 in nuclear mRNP assembly. Taken together, our integrative analysis uncovers a specific function of the RNA-binding activity of the nuclear mRNP component Npl3. This approach can be readily applied to RBPs in any RNA metabolic process.

Item Type: Journal Article
Creators:
CreatorsEmailORCIDORCID Put Code
Keil, PhilippUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Wulf, AlexanderUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Kachariya, NitinUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Reuscher, SamiraUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Huhn, KristinUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Silbern, IvanUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Altmuller, JanineUNSPECIFIEDorcid.org/0000-0003-4372-1521UNSPECIFIED
Keller, MarioUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Stehle, RalfUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Zarnack, KathiUNSPECIFIEDorcid.org/0000-0003-3527-3378UNSPECIFIED
Sattler, MichaelUNSPECIFIEDorcid.org/0000-0002-1594-0527UNSPECIFIED
Urlaub, HenningUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Straesser, KatjaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
URN: urn:nbn:de:hbz:38-692792
DOI: 10.1093/nar/gkac1206
Journal or Publication Title: Nucleic Acids Res.
Volume: 51
Number: 2
Page Range: S. 831 - 852
Date: 2023
Publisher: OXFORD UNIV PRESS
Place of Publication: OXFORD
ISSN: 1362-4962
Language: English
Faculty: Unspecified
Divisions: Unspecified
Subjects: no entry
Uncontrolled Keywords:
KeywordsLanguage
HETERONUCLEAR NMR-SPECTROSCOPY; SMALL-ANGLE SCATTERING; PHOTO-CROSS-LINKING; MASS-SPECTROMETRY; GENE-EXPRESSION; SPLICING REGULATION; PROTEIN STRUCTURES; BACKBONE DYNAMICS; BINDING PROTEIN; SR PROTEINSMultiple languages
Biochemistry & Molecular BiologyMultiple languages
URI: http://kups.ub.uni-koeln.de/id/eprint/69279

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