Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcriptome-wide mapping of human Staufen1 binding sites

ABSTRACT: Purpose: We performed RNA-Immunoprecipitation in Tandem (RIPiT) experiments against human Staufen1 (Stau1) to identify its precise RNA binding sites in a transcriptome-wide manner. To monitor the consequences of Stau1 binding in terms of target mRNA levels and ribosome occupancy, we modified the levels of endogenous Stau1 in cells by siRNA or overexpression and performed RNA-sequencing and ribosome-footprinting experiments. Staufen1 (Stau1) is a double-stranded RNA (dsRNA) binding protein implicated in mRNA transport, regulation of translation, mRNA decay and stress granule homeostasis. Here we combined RNA-Immunoprecipitation in Tandem (RIPiT) with RNase footprinting, formaldehyde crosslinking, sonication-mediated RNA fragmentation and deep sequencing to map Staufen1 binding sites transcriptome-wide. We find that Stau1 binds complex secondary structures containing multiple short helices, many of which are formed by inverted Alu elements in annotated 3'UTRs or in "strongly distal" 3'UTRs extending far beyond the canonical polyadenylation signal. Stau1 also interacts with both actively translating ribosomes and with mRNA coding sequences (CDS) and 3'UTRs in proportion to their GC-content and internal secondary structure-forming propensity. On mRNAs with high CDS GC-content, higher Stau1 levels lead to greater ribosome densities, suggesting a general role for Stau1 in modulating the ability of ribosomes to elongate through secondary structures located in CDS regions. We used HEK293 cells expressing near endogenous levels of wild-type Flag-Stau1 (65KDa isoform with an N-Terminal Flag tag). As a control we used a mutant version of Stau1 that is not functional for dsRNA binding. Formaldehyde crosslinking experiments and RNase footprinting experiments were done in two biological replicates. All RNASeq, Ribosome footprinting and PAS-Seq were done in two biological replicates.

ORGANISM(S): Homo sapiens

SUBMITTER: Alper Kucukural

PROVIDER: E-GEOD-52447 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Staufen1 senses overall transcript secondary structure to regulate translation.

Ricci Emiliano P EP Kucukural Alper A Cenik Can C Mercier Blandine C BC Singh Guramrit G Heyer Erin E EE Ashar-Patel Ami A Peng Lingtao L Moore Melissa J MJ

Nature structural & molecular biology 20131215 1

Human Staufen1 (Stau1) is a double-stranded RNA (dsRNA)-binding protein implicated in multiple post-transcriptional gene-regulatory processes. Here we combined RNA immunoprecipitation in tandem (RIPiT) with RNase footprinting, formaldehyde cross-linking, sonication-mediated RNA fragmentation and deep sequencing to map Staufen1-binding sites transcriptome wide. We find that Stau1 binds complex secondary structures containing multiple short helices, many of which are formed by inverted Alu element ...[more]

PMID: 24336223

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Project description:DEAD-box RNA helicases eIF4A and Ded1 are believed to promote translation initiation by resolving mRNA secondary structures that impede ribosome attachment at the mRNA 5’ end or subsequent scanning of the 5’UTR, but whether they perform distinct functions or act redundantly in vivo is poorly understood. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in yeast by ribosome footprint profiling. Despite similar reductions in bulk translation, inactivation of a cold-sensitive Ded1 mutant substantially reduced the TEs of >600 mRNAs, whereas inactivation of a temperature-sensitive eIF4A mutant yielded <40 similarly impaired mRNAs. The broader requirement for Ded1 did not reflect more pervasive secondary structures at low temperature, as inactivation of temperature-sensitive and cold-sensitive ded1 mutants gave highly correlated results. Interestingly, Ded1-dependent mRNAs exhibit greater than average 5’UTR length and propensity for secondary structure, implicating Ded1 in scanning though structured 5' UTRs. Reporter assays confirmed that cap- distal stem-loop insertions increase dependence on Ded1 but not eIF4A for efficient translation. While only a small fraction of mRNAs is strongly dependent on eIF4A, this dependence is significantly correlated with requirements for Ded1 and 5’UTR features characteristic of Ded1- dependent mRNAs. Our findings suggest that Ded1 is critically required to promote scanning through secondary structures within 5’UTRs; and while eIF4A cooperates with Ded1 in this function, it also promotes a step of initiation common to virtually all yeast mRNAs. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in yeast by ribosome footprint profiling.The study includes 32 samples, comprised of 16 mRNA-Seq samples and 16 ribosome footprint profiling samples, derived from biological replicates of 3 mutant strains, ded1-cs, ded1-ts and tif1-ts, and the corresponding wild-type strains. The tif1-ts mutant and its wild-type counterpart were analyzed at 30°C and 37°C.

Dataset Information

Transcriptome-wide mapping of human Staufen1 binding sites

Publications

Staufen1 senses overall transcript secondary structure to regulate translation.

Similar Datasets

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