The LARP1 La-Module recognizes both ends of TOP mRNAs

Abstract

La-Related Protein 1 (LARP1) is an RNA-binding protein that regulates the stability and translation of mRNAs encoding the translation machinery, including ribosomal proteins and translation factors. These mRNAs are characterized by a 5'-terminal oligopyrimidine (TOP) motif that coordinates their temporal and stoichiometric expression. While LARP1 represses TOP mRNA translation via the C-terminal DM15 region, the role of the N-terminal La-Module in the recognition and translational regulation of TOP mRNAs remains elusive. Herein we show that the LARP1 La-Module also binds TOP motifs, although in a cap-independent manner. We also demonstrate that it recognizes poly(A) RNA. Further, our data reveal that the LARP1 La-Module can simultaneously engage TOP motifs and poly(A) RNA. These results evoke an intriguing molecular mechanism whereby LARP1 could regulate translation and stabilization of TOP transcripts.

Keywords: LARP1; La; RRM; TOP mRNA; poly(A); translation regulation.

Figure 1.

LARP1 La-Module binds poly(A) RNA. (A) EMSA analysis of binding assays of WT La-Module with poly(A) 25-mer. (B) Quantification of three independent EMSAs of WT La-Module with poly(A) RNA. Bars are standard deviation. (C-F) EMSAs analysing WT La-Module with (C) poly(U) 20-mer, (D) poly(G) 19-mer (E) poly(C) 20-mer, and (F) poly(A) 25-mer 3ʹPO_4.

Figure 2.

LARP1 La-Module binds TOP mRNA 5ʹUTRs in a TOP motif-dependent manner. EMSAs of binding assays of WT La-Module with (A) RPS6 5ʹUTR, (B) RPS6 5ʹUTR lacking the polypyrimidine region of the TOP motif, (C) RPS6 20-mer, and (D) Capped RPS6 20-mer. (E) Quantification of three independent EMSAs of WT La-Module with indicated RNAs. Bars are standard deviation. EMSAs of binding assays of the LARP1 La-Module with (F) RPL13A 5ʹUTR and (G) RPL13A lacking the polypyrimidine region of the TOP motif

Figure 3.

An intermediate complex forms upon the addition of cold RPS6 5ʹTOP motif or RPL13A 5ʹUTR to the La-Module-poly(A) RNA complex. Competition assays conducted in the presence of poly(dI-dC) and analysed by native gel of: the La-Module-RPS6 20-mer RNA complex with cold (A) RPS6 20-mer RNA, and (B) poly(A) 25-mer RNA. Competition assays conducted in the presence of poly(dI-dC) and analysed by native gel of: the La-Module-poly(A) RNA complex with: cold (C) poly(A) 25-mer RNA, (D) RPS6 20-mer RNA, and (E) RPL13A 5ʹUTR

Figure 4.

Formation of an intermediate complex is not an artefact of aggregation or RNA base pairing, and is dependent upon an intact TOP motif. Competition assays conducted in the presence of poly(dI-dC) and analysed by native gel of the La-Module-poly(A) RNA complex with: cold (A) poly(C) RNA, (B) poly(G) RNA, (C) RPS6 5ʹUTR lacking the pyrimidine stretch of the TOP motif, and (D) RPL13A 5ʹUTR lacking the pyrimidine stretch of the TOP motif

Figure 5.

The La-Module simultaneously binds to poly(A) RNA and RPS6 TOP motif in biotin pull-down experiments. (A) Denaturing gel analysing the flow through (FT), wash (W), and pull-down streptavidin beads (B) of the indicated binding experiments. Poly(A), poly(A) 25-mer; TOP motif, RPS6 20-mer. Ethanol precipitation recovery control, loading control, RPS6 20-mer and poly(A) RNA size markers are indicated. (B) Quantification of biotin pull-down assays. Bars indicate standard error

Figure 6.

Model of potential roles for the LARP1 La-Module in TOP mRNA translation and localization to P-bodies and stress granules. (A) The LARP1 La-Module could bind the poly(A) tails and TOP motifs of different TOP transcripts to form multimeric complexes. This might contribute to the phase separation characteristic of P-bodies and stress granules, while repressing translation and minimizing transcript degradation. (B) The LARP1 La-Module could bind the poly(A) tail and TOP motif of the same TOP transcript to enhance translation (left); recognition of the 5ʹ cap by the DM15 region of LARP1 could then repress the translation of bound transcripts (right)