LARP4A recognizes polyA RNA via a novel binding mechanism mediated by disordered regions and involving the PAM2w motif, revealing interplay between PABP, LARP4A and mRNA

Abstract

LARP4A belongs to the ancient RNA-binding protein superfamily of La-related proteins (LARPs). In humans, it acts mainly by stabilizing mRNAs, enhancing translation and controlling polyA lengths of heterologous mRNAs. These activities are known to implicate its association with mRNA, protein partners and translating ribosomes, albeit molecular details are missing. Here, we characterize the direct interaction between LARP4A, oligoA RNA and the MLLE domain of the PolyA-binding protein (PABP). Our study shows that LARP4A–oligoA association entails novel RNA recognition features involving the N-terminal region of the protein that exists in a semi-disordered state and lacks any recognizable RNA-binding motif. Against expectations, we show that the La module, the conserved RNA-binding unit across LARPs, is not the principal determinant for oligoA interaction, only contributing to binding to a limited degree. Furthermore, the variant PABP-interacting motif 2 (PAM2w) featured in the N-terminal region of LARP4A was found to be important for both RNA and PABP recognition, revealing a new role for this protein–protein binding motif. Our analysis demonstrates the mutual exclusive nature of the PAM2w-mediated interactions, thereby unveiling a tantalizing interplay between LARP4A, polyA and PABP.

Crystal Structure of a Variant PAM2 Motif of LARP4B Bound to the MLLE Domain of PABPC1

“…In contrast, other LARP members, including 1, 4 and 6, interact with mRNA rather than with Pol III transcripts [ 40 , 41 , 42 , 43 ]. Moreover, with the exception of LARP6, these cytoplasmic LARP proteins all harbour PAM2 motifs, and likely bind PABPC1 [ 44 , 45 , 46 , 47 ]. Within this group, LARP4 and LARP4B stand apart, as they harbor a variant PAM2 motif close to their N-termini (termed PAM2w), in which a conserved Tyrosine residue is replaced by Tryptophan [ 48 ].…”

Single molecule poly(A) tail-seq shows LARP4 opposes deadenylation throughout mRNA lifespan with most impact on short tails

“…( D ) Top: schematic of LARP4 showing its two PABP-interaction motifs, PAM2w and PBM, the La-module comprised of LaM (La motif) and RRM-L4, and the RACK-1 interaction region (RIR). The N-terminal region (NTR) which is responsible for poly(A)-binding ( Cruz-Gallardo et al, 2019 ), is also indicated. Middle: Northern blot after co-transfection of 1 kb AT-rich DNA or pUC19 and various LARP4 constructs indicated above the lanes.…”

“…LARP4 is a mRNA stabilizing protein that can bind to poly(A) via its N-terminal region (NTR) which involves the PAM2w, and also binds PABP via another motif ( Cruz-Gallardo et al, 2019 ; Yang et al, 2011 ). We developed a single molecule mRNA sequencing method for measuring and quantifying PAT lengths transcriptome-wide to study effects of LARP4 on poly(A) metabolism.…”

“…The data here leave open the possibility that LARP4 may directly protect RNA 3’ ends, and also support a model in which it stabilizes PABP on short PATs to shield them from deadenylases. In this model, decreased PABP-PAT stability would be compensated by interactions with LARP4 ( Cruz-Gallardo et al, 2019 ; Mattijssen et al, 2017 ; Yang et al, 2011 ). Such interactions are likely intricate as LARP4 can employ two motifs for PABP binding including by its unique PAM2w which can also be used to alternatively bind poly(A) ( Cruz-Gallardo et al, 2019 ).…”

“…LARP4, which is also known as LARP4A directly binds poly(A) RNA and PABP ( Cruz-Gallardo et al, 2019 ; Yang et al, 2011 ). LARP4 is a short-lived protein whose accumulation levels can span a wide range due in part to control by independent instability elements located in the coding region and in the 3’UTR of its mRNA ( Mattijssen et al, 2017 ; Mattijssen and Maraia, 2016 ).…”