Signal sequence- and translation-independent mRNA localization to the endoplasmic reticulum.
The process of mRNA localization typically utilizes cis-targeting elements and trans-recognition factors to direct the compartmental organization of translationally suppressed mRNAs. mRNA localization to the endoplasmic reticulum (ER), in contrast, occurs via a co-translational, signal sequence/signal recognition particle (SRP)-dependent mechanism. We have utilized cell fractionation/cDNA microarray analysis, shRNA-mediated suppression of SRP expression, and mRNA reporter construct studies to define the role of the SRP pathway in ER-directed mRNA localization. Cell fractionation studies of mRNA partitioning between the cytosol and ER demonstrated the expected enrichment of cytosolic/nucleoplasmic protein-encoding mRNAs and secretory/integral membrane protein-encoding mRNAs in the cytosol and ER fractions, respectively, and identified a subpopulation of cytosolic/nucleoplasmic protein-encoding mRNAs in the membrane-bound mRNA pool. The latter finding suggests a signal sequence-independent pathway of ER-directed mRNA localization. Extending from these findings, mRNA partitioning was examined in stable SRP54 shRNA knockdown HeLa cell lines. shRNA-directed reductions in SRP did not globally alter mRNA partitioning patterns, although defects in membrane protein processing were observed, further suggesting the existence of multiple pathways for mRNA localization to the ER. ER localization of GRP94-encoding mRNA was observed when translation was disabled by mutation of the start codon/insertion of a 5'UTR stem-loop structure or upon deletion of the encoded signal sequence. Combined, these data indicate that the mRNA localization to the ER can be conferred independent of the signal sequence/SRP pathway and suggest that mRNA localization to the ER may utilize cis-encoded targeting information.
Pyhtila
Brook
B
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Zheng
Tianli
T
Lager
Patrick J
PJ
Keene
Jack D
JD
Reedy
Mary C
MC
Nicchitta
Christopher V
CV
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D018271:Signal Recognition Particle / Q000235:genetics* / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D017931:DNA Primers; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D018271:Signal Recognition Particle; C053387:endoplasmin