Intramural Research Program, <italic>Eunice Kennedy Shriver</italic> National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States.
Commissioned Corps, U.S. Public Health Service, Rockville, United States.
Elife. 2020 Aug 3;9:e59186. doi: 10.7554/eLife.59186.
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 consistent with greater PABP dissociation in the absence of LARP4. Thus, LARP4 shapes PAT profiles throughout mRNA lifespan with impact on mRNA decay at short lengths known to sensitize PABP dissociation in response to deadenylation machinery.
LARP4 蛋白直接结合多聚腺苷酸（poly(A)）和多聚腺苷酸结合蛋白（PABP）。研究表明，LARP4 通过防止脱腺苷酸化，促进多聚腺苷酸尾（PAT）的延长和单个 mRNA 的稳定（Mattijssen 等人，2017）。我们开发了一种核苷酸分辨率的全转录组单分子 SM-PAT-seq 方法。该方法揭示了 LARP4 对人类和小鼠 mRNA 的广泛 PAT 长度的影响，这些 mRNA 来自 LARP4 敲除（KO）和对照细胞。LARP4 的影响在长 PAT mRNA 上很明显，但在 30-75 个核苷酸处更为明显。我们还分析了全转录组范围内和大约 200 个免疫反应 mRNA 的 PAT 衰减时间过程。这表明，在 PATs<75 个核苷酸的 KO 细胞中，脱腺苷酸化加速，与缺乏 LARP4 时 PABP 解离更为一致。因此，LARP4 在 mRNA 寿命的整个过程中塑造 PAT 分布，在已知对脱腺苷酸化机制的 PABP 解离敏感的短长度上对 mRNA 衰变产生影响。
