Human La Function in Expression of Coding Transcripts
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The La protein, also referred to as Sjogrens Syndrome antigen B (SSB), is an RNA-binding phosphoprotein first identified as an auto-antigen in patients suffering from Sjogrens syndrome and systemic lupus erythematosus. La proteins are present and indispensable in nearly all eukaryotes and exhibit conserved functions in RNA metabolism. Nuclear La is involved in facilitating the processing and maturation of RNA polymerase III transcripts by binding to the UUU-3'OH trailer in a sequence specific manner. Cytoplasmic La proteins are associated with promoting cap-independent translation from the internal ribosome entry sites (IRESs) of several cellular and viral coding RNAs. In this study, we investigated the molecular mechanisms by which La interacts with coding transcripts. We show that in addition to a sequence specific UUU-3'OH binding mode, human La exhibits both a sequence specific and length dependent poly(A) binding mode mapped to the canonical winged helix face of the eponymous La motif. We demonstrate that cytoplasmic La engages poly(A) RNA in human cells, and La promotion of translation from the cyclin D1 IRES occurs in competition with the cytoplasmic poly(A) binding protein (PABP). During viral infection, tumor progression, and certain forms of cellular stress, the predominantly nuclear hLa protein has been shown to translocate to the cytoplasm where it functions as an IRES trans-acting factor (ITAF), controlling cap-independent translation initiation of several cellular IRES-containing mRNAs involved in stress responses, signifying an important role for hLa during stress. We aim to understand the cellular conditions during which La interacts with its cytoplasmic substrates and identify novel RNA substrates of La. We show that cellular stress induced by clotrimazole results in translocation of hLa from the nucleus to the cytoplasm. Using polysome analysis and qPCR, we also show that La translocation into cytoplasm is concurrent with increased association of La with actively translating messages. Using iCLIP, we identify novel hLa associated targets. Taken together, these results suggest that hLa is trafficked into the cytoplasm in response to cellular stress in order to associate with IRES containing messages, and this interaction may occur through contacts made via a novel binding mode with the poly(A) tail.