RNA Chaperone Activity of Human La Protein Is Mediated by Variant RNA Recognition Motif

Amir R Naeeni,Maria R Conte,Mark A Bayfield

doi:10.1074/jbc.m111.276071

Amir R Naeeni, Maria R Conte + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m111.276071

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Abstract

La proteins are conserved factors in eukaryotes that bind and protect the 3′ trailers of pre-tRNAs from exonuclease digestion via sequence-specific recognition of UUU-3′OH. La has also been hypothesized to assist pre-tRNAs in attaining their native fold through RNA chaperone activity. In addition to binding polymerase III transcripts, human La has also been shown to enhance the translation of several internal ribosome entry sites and upstream ORF-containing mRNA targets, also potentially through RNA chaperone activity. Using in vitro FRET-based assays, we show that human and Schizosaccharomyces pombe La proteins harbor RNA chaperone activity by enhancing RNA strand annealing and strand dissociation. We use various RNA substrates and La mutants to show that UUU-3′OH-dependent La-RNA binding is not required for this function, and we map RNA chaperone activity to its RRM1 motif including a noncanonical α3-helix. We validate the importance of this α3-helix by appending it to the RRM of the unrelated U1A protein and show that this fusion protein acquires significant strand annealing activity. Finally, we show that residues required for La-mediated RNA chaperone activity in vitro are required for La-dependent rescue of tRNA-mediated suppression via a mutated suppressor tRNA in vivo. This work delineates the structural elements required for La-mediated RNA chaperone activity and provides a basis for understanding how La can enhance the folding of its various RNA targets.

Highlights

La proteins are conserved throughout most eukaryotes and are highly expressed RNA binding factors with important functions in the processing and metabolism of a variety of RNA targets [1]
To examine the importance of the canonical RNA recognition motif (RRM), the RRM1 ␣3-helix, and linker region further, we tested RRM1 without the La motif but including successively greater sections of the region between amino acids 187 and 235 in the assays. We found that these RRM1 mutants acquired modest strand annealing activity with successively greater C-terminal extensions, consistent with the RRM, ␣3-helix, and adjacent linker representing the necessary elements for strand association activity
In addition to a hypothesized function in the folding of misfolded pre-tRNAs ending in UUU-3ЈOH, La proteins have been hypothesized to function as an RNA chaperone for mRNAs lacking this motif by enhancing the correct folding of complex 5Ј-UTR structures required for optimal translation

Summary

Introduction

La proteins are conserved throughout most eukaryotes and are highly expressed RNA binding factors with important functions in the processing and metabolism of a variety of RNA targets [1]. Conserved RNA Chaperone Activities of La Proteins—To assay the strand annealing and dissociation activities of purified hLa, we first used a modified, 33-nucleotide top strand containing the 21-nucleotide region of complimentarity followed by a 12-nucleotide trailer ending with UUUU-3ЈOH (21F Cy5 Trailer UUUU; supplemental Table S1 and Fig. 2A), because we had previously shown this trailer sequence binds hLa with high affinity using the UUU-3ЈOH-dependent binding mode [14].

Results

Conclusion