Substrate Specificity of the TRAMP Nuclear Surveillance Complexes

During nuclear surveillance, the RNA exosome functions together with the TRAMP complexes, which include the RNA helicase Mtr4 together with a RNA binding protein (Air1 or Air2) and a poly(A) polymerase (Trf4 or Trf5). To better determine how RNA substrates are targeted, we analyzed protein and RNA interactions by TRAMP components. Mass spectrometry identified three distinct TRAMP complexes formed in vivo. These complexes preferentially assemble on different classes of transcripts. Unexpectedly, on many substrates, including pre-rRNAs and pre-mRNAs, specificity was apparently conferred by Trf4 and Trf5. Clustering of mRNAs by TRAMP association showed co-enrichment for mRNAs with functionally related products, supporting the significance of surveillance in regulating gene expression. Comparison of the binding sites of TRAMP components with multiple nuclear RNA binding proteins revealed preferential colocalization of subsets of factors. On pre-mRNAs, ∆trf5 deletion caused reduced Mtr4 binding and RNA accumulation of Trf5 top targets.在核RNA监视过程中，RNA外切体（RNA exosome）会与TRAMP复合物（TRAMP complexes）协同发挥功能；TRAMP复合物包含RNA解旋酶（RNA helicase）Mtr4、RNA结合蛋白（RNA binding protein，Air1或Air2）以及多聚腺苷酸聚合酶（poly(A) polymerase，Trf4或Trf5）。为更清晰地阐明RNA底物的靶向识别机制，本研究通过TRAMP组分分析了蛋白质与RNA的相互作用。质谱（mass spectrometry）鉴定出体内形成了三种不同的TRAMP复合物，这类复合物会优先组装于不同类别的转录本（transcript）之上。出乎意料的是，在包括核糖体RNA前体（pre-rRNA）和信使RNA前体（pre-mRNA）在内的诸多底物中，靶向特异性显然由Trf4与Trf5赋予。通过TRAMP结合特征对信使RNA（mRNA）进行聚类分析后发现，编码功能相关产物的mRNA呈现共富集现象，这证实了RNA监视通路在基因表达调控中的重要意义。将TRAMP组分的结合位点与多种核RNA结合蛋白的结合位点进行比对后发现，特定亚群的核因子存在优先共定位现象。在信使RNA前体（pre-mRNA）上，trf5基因缺失会导致Mtr4的结合水平降低，同时也会减少Trf5顶级靶标的RNA积累量。