Examining SRP pathway function in mRNA localization to the endoplasmic reticulum

Signal recognition particle (SRP) pathway function in secretory/membrane protein translocation across the endoplasmic reticulum (ER) is well-established; its role in mRNA localization to the ER in mammalian cells remains largely unexplored. We address this question in SRP receptor (SR) knockout mammalian cell lines. SRPRB KO cells were generated by CRISPR editing. SRPRB KO resulted in profound destabilization of SRα with siRNA silencing of SRPRA in SRPRB KO cells yielding SR KO. Steady-state mRNA composition and ER-enrichments were not disrupted by loss of SR. SR function in the ER localization of newly exported mRNAs was examined by 4-thiouridine (4SU) pulse-chase/4SU-seq and found to be SR-independent. Under conditions of translation initiation inhibition, the ER was the default localization site for newly exported mRNAs. These data demonstrate that mRNA localization to the ER can be uncoupled from SRP pathway function and reopen questions regarding the mechanism of mRNA localization to the ER. There are two components to this study. In the first component, RNA-seq studies were performed on the cytosolic and ER-bound mRNAs from parental, SRPRB knockout, SRPRB knockout + SRPRA siRNA knockdown HeLa cells, to examine the steady-state transcriptomic response to loss of SRP receptor expression. In a second set of studies, the localization of newly exported mRNAs to the cytosol and ER compartments of HeLa Cas9 and HeLa SRPRB knockout/SRPRA siRNA knockdown cells was study by biosynthetic pulse chase. Here, 4-thiouridine (4SU) transcriptional labeling was used to identify newly transcribed and exported mRNAs. 4SU pulse-chase studies were coupled with cell fractionation to identify the subcellular localization site (cytoplasm or endoplasmic reticulum) of 4SU-labeled mRNAs. 4SU mRNAs were isolated following total RNA isolation, 4SU-directed biotinylation, streptavidin-magnetic bead isolation, and oligo-DT selection of poly(A) mRNAs. Time course studies at five time points (0, 15 min, 30 min, 45 min, 60 min) were performed to determine relative nuclear export rates. Subsequently, 4SU transcriptional pulse-labeling studies were performed to examine the effects of translational inhibition on the localization of newly exported mRNAs. In these experiments, cells were treated with either vehicle (control)), DTT (to activate the UPR), or harringtonine, an inhibitor of translation initiation, and 4SU mRNAs captured at the 45 min time point. In a third set of experiments, the role of the SRP pathway in the trafficking of newly exported mRNAs to the cytosol and ER was examined by 4SU-seq analyses in parental vs SR KO cells. To correct for background, parallel experiments were performed where 4SU was omitted and the samples processed as per the experimental (+4SU) conditions and sequenced. Experiments were performed in biological duplicate or triplicate, 4SU-mRNA distributions were assayed by qRT-PCR of marker genes, and samples processed for deep sequencing. A thiolated spike-in control poly(A) mRNA, comprising a Bacillus subtilis sequence, was included for recovery normalization.