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Vigilin/HDLBP promotes translation of endoplasmic reticulum-targeted mRNAs (tRNA ncRNA-seq)

ABSTRACT: The biological role of RNA-binding proteins (RBPs) in the secretory pathway and their contribution to the recognition and co-translational targeting of ER-localized mRNAs is not well established. In this work we used biochemical, transcriptomic and proteomic approaches to delineate the role of human HDLBP/vigilin. PAR-CLIP analysis revealed that HDLBP directly and specifically interacted with more than 80% of all expressed ER-localized mRNAs. Interestingly, the binding to the coding sequence was most prominent for ER-localized mRNAs, while cytosolic mRNAs showed higher binding in the 3’UTR. HDLBP crosslinked strongly to long CU-rich motifs that resided more frequently in coding sequences of ER-localized but not in cytosolic mRNAs. This indicated that the primary sequence composition determines the basis for HDLBP binding specificity and its multivalent interactions with ER-bound mRNAs. PAR-CLIP analysis also revealed direct interactions of HDLBP with the RNA components of the translational apparatus, while in vivo proximity proteomics detected proteins involved in translation and components of the signal recognition particle (SRP). Functional studies using CRISPR-Cas9 HDLBP knockout cell lines in combination with ribosome profiling, pSILAC, and luciferase assays showed decreased translation efficiency of HDLBP target mRNAs, impaired protein synthesis and secretion in the knockout conditions. Finally, HDLBP absence resulted in decrease of in vivo lung tumor formation. These results highlight a general function for HDLBP in the translation of ER -localized mRNAs via the secretory pathway and discover its relevance for cell proliferation and tumor progression.

ORGANISM(S): Homo sapiens

PROVIDER: GSE148261 | GEO | 2022/02/04

REPOSITORIES: GEO

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Vigilin/HDLBP promotes translation of endoplasmic reticulum-targeted mRNAs (HDLBP PAR-CLIP)

Project description:The biological role of RNA-binding proteins (RBPs) in the secretory pathway and their contribution to the recognition and co-translational targeting of ER-localized mRNAs is not well established. In this work we used biochemical, transcriptomic and proteomic approaches to delineate the role of human HDLBP/vigilin. PAR-CLIP analysis revealed that HDLBP directly and specifically interacted with more than 80% of all expressed ER-localized mRNAs. Interestingly, the binding to the coding sequence was most prominent for ER-localized mRNAs, while cytosolic mRNAs showed higher binding in the 3’UTR. HDLBP crosslinked strongly to long CU-rich motifs that resided more frequently in coding sequences of ER-localized but not in cytosolic mRNAs. This indicated that the primary sequence composition determines the basis for HDLBP binding specificity and its multivalent interactions with ER-bound mRNAs. PAR-CLIP analysis also revealed direct interactions of HDLBP with the RNA components of the translational apparatus, while in vivo proximity proteomics detected proteins involved in translation and components of the signal recognition particle (SRP). Functional studies using CRISPR-Cas9 HDLBP knockout cell lines in combination with ribosome profiling, pSILAC, and luciferase assays showed decreased translation efficiency of HDLBP target mRNAs, impaired protein synthesis and secretion in the knockout conditions. Finally, HDLBP absence resulted in decrease of in vivo lung tumor formation. These results highlight a general function for HDLBP in the translation of ER -localized mRNAs via the secretory pathway and discover its relevance for cell proliferation and tumor progression.

2022-02-04 | GSE148259 | GEO

Vigilin/HDLBP promotes translation of endoplasmic reticulum-targeted mRNAs (WT vs HDLBP Ribo-seq + RNA-seq)

2022-02-04 | GSE148258 | GEO

Vigilin/HDLBP promotes translation of endoplasmic reticulum-targeted mRNAs (Cell fractionation RNA-seq)

2022-02-04 | GSE148260 | GEO

Vigilin/HDLBP promotes translation of endoplasmic reticulum-targeted mRNAs (A549 HDLBP KO and WT)

2022-02-04 | GSE150691 | GEO

Vigilin/HDLBP promotes translation of endoplasmic reticulum-targeted mRNAs

Project description:Nascent peptide chain targeting to the endoplasmic reticulum (ER) membrane is required for correct localization and efficient translation of membrane-bound and secreted proteins. However, little is known about the contribution of RNA-binding proteins (RBPs) to the recognition, localization and translation of ER-localized mRNAs. In this work we used biochemical, transcriptomic and proteomic approaches to delineate the role of human HDLBP. PAR-CLIP analysis revealed that HDLBP directly and specifically interacted with more than 80% of all expressed ER-localized mRNAs. Interestingly, the binding to the coding sequence was most prominent for ER-localized mRNAs, while cytosolic mRNAs showed higher binding in the 3’UTR. HDLBP crosslinked strongly to long CU-rich motifs that resided more frequently in coding sequences of ER-localized but not in cytosolic mRNAs. This indicated that the primary sequence composition determines the basis for HDLBP binding specificity and its multivalent interactions with ER-bound mRNAs. PAR-CLIP analysis also revealed direct interactions of HDLBP with the RNA components of the translational apparatus, while in vivo proximity proteomics detected proteins involved in translation and components of the signal recognition particle (SRP). Functional studies using CRISPR-Cas9 HDLBP knockout cell lines in combination with ribosome profiling, pSILAC, and luciferase assays showed decreased translation efficiency of HDLBP target mRNAs, impaired protein synthesis and secretion in the knockout conditions. Finally, HDLBP absence resulted in decrease of lung tumor formation capacity in vivo. These results highlight a general function for HDLBP in the translation of ER -localized mRNAs via the secretory pathway and discover its relevance for cell profileration and tumor progression

2022-05-09 | PXD018316 | Pride

Vigilin/HDLBP promotes translation of endoplasmic reticulum-targeted mRNAs

2022-06-09 | PXD018313 | Pride

Vigilin/HDLBP promotes translation of endoplasmic reticulum-targeted mRNAs (HDLBP PAR-CLIP)

Project description:Vigilin/HDLBP promotes translation of endoplasmic reticulum-targeted mRNAs (HDLBP PAR-CLIP)

| PRJNA623611 | ENA

Identification of LIN28-bound mRNAs reveals features of target recognition and regulation

Project description:The conserved human LIN28 RNA-binding proteins function in development, maintenance of pluripotency and oncogenesis. We used PAR-CLIP and a newly developed variant of this method, iDo-PAR-CLIP, to identify LIN28B targets as well as sites bound by the individual RNA binding domains of LIN28B in the human transcriptome at nucleotide resolution. The position of target binding sites reflected the known structural relative orientation of individual LIN28B binding domains, validating iDo-PAR-CLIP. Our data suggest that LIN28B directly interacts with most expressed mRNAs and members of the let-7 microRNA family. The Lin28 binding motif detected in pre-let-7 was enriched in mRNA sequences bound by LIN28B. Upon LIN28B knock down, cell proliferation and the cell cycle were strongly impaired. Quantitative shotgun proteomics of LIN28B depleted cells revealed significant reduction of protein synthesis from its RNA targets that function in translation, mRNA splicing and cell cycle control. Computational analyses provided evidence that the strength of protein synthesis reduction correlated with the location of LIN28B binding sites within target transcripts. We used PAR-CLIP and a newly developed variant of this method, iDo-PAR-CLIP, to identify LIN28B targets as well as sites bound by the individual RNA binding domains of LIN28B in the human transcriptome at nucleotide resolution.

2013-06-25 | E-GEOD-46908 | biostudies-arrayexpress

Cotranslational signal independent SRP preloading during membrane targeting

Project description:Cotranslational targeting into the endoplasmic reticulum (ER) by the Signal Recognition Particle (SRP) is a key event determining polypeptide fate in eukaryotic cells. Here, we globally define the principles and mechanisms of SRP binding and ER targeting in vivo. Cotranslational targeting through SRP is the dominant route into the ER for all secretory proteins, regardless of targeting signal characteristics. Cytosolic SRP functions in a pioneer translation round that builds a membrane-resident mRNAs pool, explaining how low SRP levels suffice for the secretory load. SRP does not induce an elongation arrest; consequently, kinetic competition between targeting and translation elongation dictates which substrates are translocated post-translationally. Unexpectedly, SRP binds most secretory ribosomal complexes before targeting signals are synthesized. We show non-coding mRNA elements can promote signal-independent SRP pre-recruitment. Our study defines the complex kinetic interplay between elongation and determinants in the polypeptide and mRNA modulating SRP-substrate selection and membrane targeting in vivo. Ribosome profiling (RiboSeq) and RNA-seq of subcellular fractions of ribosomes. Soluble and membrane bound ribosomes are separated by centrifugation, and SRP-bound ribosomes are immunoprecipitated from the soluble fraction. Polysomes and monosomes are separated by sucrose gradient ultracentrifugation.

2016-08-03 | E-GEOD-74393 | biostudies-arrayexpress

Ribosome footprinting in the cytosol and endoplasmic reticulum

Project description:In eukaryotic cells, the spatial regulation of protein expression is frequently conferred through the coupling of mRNA localization and the local control of translation. mRNA localization to the endoplasmic reticulum (ER) is a prominent example of such regulation and serves a ubiquitous role in segregating the synthesis of secretory and integral membrane proteins to the ER. Recent genomic and biochemical studies have now expanded this view to suggest a role for the ER in global protein synthesis. We have utilized cell fractionation and ribosome profiling to obtain a genomic survey of the subcellular organization of mRNA translation and report that ribosomal loading of mRNAs, a proxy for mRNA translation, is biased to the ER. Notably, ER-associated mRNAs encoding both cytosolic and topogenic signal-encoding proteins display similar ribosome loading densities, suggesting that ER-associated ribosomes serve a global role in mRNA translation. We propose that the partitioning of mRNAs and their translation between the cytosol and ER compartments may represent a novel mechanism for the post-transcriptional regulation of gene expression. HEK293 cells were fractionated between the cytosol and endoplasmic reticulum. Within each fraction, ribosome footprints were generated and sequenced. In parallel, total mRNA was sequenced.

2011-12-20 | E-GEOD-31539 | biostudies-arrayexpress

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