Screening of the targeted mRNAs of ER-anchored PARN by transcriptome sequencing.
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ABSTRACT:
PROVIDER: PRJNA492131 | ENA |
REPOSITORIES: ENA
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Project description:The goals of this study are to identify the potential targets and then elucidate the function of ER-anchored PARN. After overexpressing the empty vector, PARN(wild type) or D28A(the enzyme-dead mutant of PARN) in HEK-293T cell respectively and the following fractionation of the cytosol and ER fraction, we performed the high-throughput RNA sequencing for each fraction. After comparing the abundance of detected transcripts in different samples we found ER-anchored PARN only affects a small subset of mRNAs in HEK-293T cell.
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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.
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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
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Project description:Almost all cellular mRNAs terminate in a 3M-bM-^@M-^Y poly(A) tail, the removal of which can induce both translational silencing and mRNA decay. Mammalian cells encode many poly(A)-specific exoribonucleases but their individual roles are poorly understood. Here, we undertook an analysis of the role of PARN deadenylase in mouse myoblasts using global measurements of mRNA decay rates. Our results reveal that a discrete set of mRNAs exhibit altered mRNA decay as a result of PARN depletion and that stabilization is associated with increased poly(A) tail length and translation. We determined that stabilization of mRNAs does not generally result in their increased abundance supporting the idea that mRNA decay is coupled to transcription. Importantly, PARN knockdown has wide ranging effects on gene expression that specifically impact the extracellular matrix and cell migration. Finally, although PARN has its own unique target transcripts it also influences some genes whose expression is modulated by other deadenylases. In order to investigate the role of PARN deadenyl on mRNA decay in mouse muscle cells , C2C12 cells were transfected by shRNA knockdown of PARN and treated with actinomycin D to inhibit transcription. Total RNA was isolated from three replicates at 0, 15, 30, 60, 120 and 240 minutes. The CTRL cell line stably transfected with LKO1 vector was described previously (GSE21233).
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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.
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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.
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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.
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Project description:Almost all cellular mRNAs terminate in a 3’ poly(A) tail, the removal of which can induce both translational silencing and mRNA decay. Mammalian cells encode many poly(A)-specific exoribonucleases but their individual roles are poorly understood. Here, we undertook an analysis of the role of PARN deadenylase in mouse myoblasts using global measurements of mRNA decay rates. Our results reveal that a discrete set of mRNAs exhibit altered mRNA decay as a result of PARN depletion and that stabilization is associated with increased poly(A) tail length and translation. We determined that stabilization of mRNAs does not generally result in their increased abundance supporting the idea that mRNA decay is coupled to transcription. Importantly, PARN knockdown has wide ranging effects on gene expression that specifically impact the extracellular matrix and cell migration. Finally, although PARN has its own unique target transcripts it also influences some genes whose expression is modulated by other deadenylases.
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