An interdomain bridge influences RNA binding of the human La protein.

La proteins are RNA chaperones that perform various functions depending on distinct RNA-binding modes and their subcellular localization. In the nucleus, they help process UUU-3'OH-tailed nascent RNA polymerase III transcripts, such as pre-tRNAs, whereas in the cytoplasm they contribute to translation of poly(A)-tailed mRNAs. La accumulation in the nucleus and cytoplasm is controlled by several trafficking elements, including a canonical nuclear localization signal in the extreme C terminus and a nuclear retention element (NRE) in the RNA recognition motif 2 (RRM2) domain. Previous findings indicate that cytoplasmic export of La due to mutation of the NRE can be suppressed by mutations in RRM1, but the mechanism by which the RRM1 and RRM2 domains functionally cooperate is poorly understood. In this work, we use electromobility shift assays (EMSA) to show that mutations in the NRE and RRM1 affect binding of human La to pre-tRNAs but not UUU-3'OH or poly(A) sequences, and we present compensatory mutagenesis data supporting a direct interaction between the RRM1 and RRM2 domains. Moreover, we use collision-induced unfolding and time-resolved hydrogen-deuterium exchange MS analyses to study the conformational dynamics that occur when this interaction is intact or disrupted. Our results suggest that the intracellular distribution of La may be linked to its RNA-binding modes and provide the first evidence for a direct protein-protein interdomain interaction in La proteins.

Human La binds mRNAs through contacts to the poly(A) tail.

In addition to a role in the processing of nascent RNA polymerase III transcripts, La proteins are also associated with promoting cap-independent translation from the internal ribosome entry sites of numerous cellular and viral coding RNAs. La binding to RNA polymerase III transcripts via their common UUU-3'OH motif is well characterized, but the mechanism of La binding to coding RNAs is poorly understood. Using electromobility shift assays and cross-linking immunoprecipitation, we show that in addition to a sequence specific UUU-3'OH binding mode, human La exhibits a sequence specific and length dependent poly(A) binding mode. We demonstrate that this poly(A) binding mode uses the canonical nucleic acid interaction winged helix face of the eponymous La motif, previously shown to be vacant during uridylate binding. We also show that cytoplasmic, but not nuclear La, engages poly(A) RNA in human cells, that La entry into polysomes utilizes the poly(A) binding mode, and that La promotion of translation from the cyclin D1 internal ribosome entry site occurs in competition with cytoplasmic poly(A) binding protein (PABP). Our data are consistent with human La functioning in translation through contacts to the poly(A) tail.