Premium
Ribosomal Protein L5 Plays a Moonlighting Role in HAC1 mRNA
Author(s) -
Ashish Anshu Fnu,
Dey Madhusudan
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.lb216
Subject(s) - rna , microbiology and biotechnology , intron , chemistry , biochemistry , ribosome , biology , gene
The unfolded protein response (UPR) pathway is a conserved pathway from yeast to human, which maintains cellular protein homeostasis. An endoplasmic reticulum (ER)‐resident RNase Ire1 initiates the UPR in yeast Saccharomyces cerevisiae by cleaving out an inhibitory intron from the HAC1 mRNA, thus generating a new open reading frame that yields Hac1 protein. Hac1 is a transcription factor that activates the expression of the UPR genes. For effective cleavage and processing, HAC1 mRNA assembles around the Ire1 protein, which requires a 3'‐bipartite element (3'BE) located at the 3'‐untranslated leader. To understand the inner mechanisms of the 3'BE‐mediated HAC1 ‐mRNAIre1 protein assembly, we expressed in vivo a 3'BE‐RMB mini RNA consisting of 48 nucleotides of the BE mRNA and 43 nucleotides of an RNA aptamer mimicking biotin (RNA m imic of b iotin, RMB). Uracils of the 3'BE‐RMB mini RNA were labeled with a thiol group while growing yeast cells in the presence of 4‐thiouridine (4sU). The 4sU‐labeled RNA and bound proteins were photo‐crosslinked and immobilized on streptavidin‐agarose. The 3'BE‐RMB mini RNA bound proteins were identified by SDS‐PAGE followed by LC/MALDI mass spectrophotometry. We identified two S1 RNA‐binding domain‐containing proteins: the ribosomal protein L5 (RpL5) and the a‐subunit of initiation factor eIF2 (eIF2a). An in vitro electrophoretic mobility shift assay showed that RpL5, but not eIF2a, specifically bound to the synthetic 3'BE RNA. Furthermore, we found that targeted mutations in the RPL5 gene reduced the normal yeast growth, but increased the ER stress resistance. These results suggest that, apart from ribosome biogenesis, RpL5 plays a moonlighting role in the UPR signaling while regulating HAC1 mRNA processing.