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

Abstract

La-related protein 4 (LARP4) directly binds both poly(A) and poly(A)-binding protein (PABP). LARP4 was shown to promote poly(A) tail (PAT) lengthening and stabilization of individual mRNAs presumably by protection from deadenylation (Mattijssen et al., 2017). We developed a nucleotide resolution transcriptome-wide, single molecule SM-PAT-seq method. This revealed LARP4 effects on a wide range of PAT lengths for human mRNAs and mouse mRNAs from LARP4 knockout (KO) and control cells. LARP4 effects are clear on long PAT mRNAs but become more prominent at 30-75 nucleotides. We also analyzed time courses of PAT decay transcriptome-wide and for ~200 immune response mRNAs. This demonstrated accelerated deadenylation in KO cells on PATs <75 nucleotides and phasing features consistent with greater PABP dissociation in the absence of LARP4. Thus, LARP4 shapes PAT profiles throughout mRNA lifespan and with impact on mRNA decay at short lengths known to sensitize PABP dissociation in response to deadenylation machinery.

Novel insights into the molecular mechanisms underlying risk of colorectal cancer from smoking and red/processed meat carcinogens by modeling exposure in normal colon organoids

“…From our WGCNA, we identified candidate GWAS-related genes LARP4 and PIP4K2C as respective hub genes for the plum4 and lightsteelblue modules. In other model systems, LARP4 has been implicated in mRNA stabilization [ 44 ]. Consistent with this, the plum4 module was enriched for numerous regulatory processes including posttranscriptional regulation of gene expression.…”

RNA-protein interactome at the Hepatitis E virus internal ribosome entry site

“…EEF2 (eukaryotic elongation factor 2) is a GTP binding translation elongation factor, which plays an essential role in translation by promoting GTP dependent translocation of the ribosome (66). LARP4 binds to the poly-A tract of mRNA, associates with the 40S ribosomal subunit as well as polysomes and plays a role in translation regulation by preventing deadenylation at poly-A tail (67)(68)(69). Presence of these factors in the HEV IRESl RNA-associated protein complex suggests their potential role(s) in regulating the HEV IRESl-mediated translation.…”

CLT-seq as a universal homopolymer-sequencing concept reveals poly(A)-tail-tuned ncRNA regulation

“…(Houseley et al 2006) Even so, the transcriptome-wide analyses of poly(A)-tail length started only when the TAIL-seq (Chang et al 2014) and PAL-seq (Subtelny et al 2014) methods broke through the homopolymer base-calling technical limits. In recent years the Nanopore technology (Garalde et al 2018) and PacBio system (Legnini et al 2019;Liu et al 2019;Mattijssen et al 2020) also proved to overcome homopolymer-reading barriers. However, all such methods for the high-quality homopolymer reading must rely on an elaborate sequencer-dependent library-preparing plan that inevitably introduces biases in sequencing.…”

“…(Chang et al 2014; Lim et al 2016; Lima et al 2017) An internal standard and calibration curve method in PAL-seq was utilized to estimate poly(A)-tail length. (Subtelny et al 2014; Eisen et al 2020) Further advancements in long-read sequencing by Nanopore(Garalde et al 2018; Nicholson-Shaw et al 2022) and PacBio(Legnini et al 2019; Liu et al 2019; Mattijssen et al 2020; Long et al 2021) also handled homopolymeric sequences through repeatedly correcting reading-errors. All these methods have indeed expanded our understanding of the dynamic nature of the mRNA poly(A) tail.…”