The exosome component Rrp6 is required for RNA polymerase II termination at specific targets of the Nrd1-Nab3 pathway.
The exosome component Rrp6 is required for RNA polymerase II termination at specific targets of the Nrd1-Nab3 pathway.
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DOI：
10.1371/journal.pgen.1004999
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发表时间：
2015
期刊：
影响因子：
4.5
通讯作者：
Mosley AL
中科院分区：
文献类型：
--
作者：
Fox MJ;Gao H;Smith-Kinnaman WR;Liu Y;Mosley AL
外泌体及其核特异性亚基Rrp 6形成3 '-5'外切核酸酶复合物，其调节RNA生物学的多个方面，包括多种非编码RNA（ncRNA）和不稳定转录物的3'末端加工和降解。核外泌体的已知靶点包括短（<1000 bp）RNAPII转录物，如小的非编码RNA（snRNA）、隐蔽不稳定转录物（CUT）和一些稳定的未注释转录物（SUT），这些转录物通过Nrd 1、Nab 3和Sen 1（NNS）依赖性机制终止。NNS依赖性终止与酵母中Rrp 6/外泌体的RNA 3'端加工和/或降解偶联。最近的工作表明，Nrd 1是必要的转录监视，调节启动子方向性和抑制反义转录独立，或之前，Rrp 6的活动。目前还不清楚Rrp 6是否直接参与终止;然而，Rrp 6已经涉及ncRNA转录物（包括CUT）的3'端加工和降解。为了确定Rrp 6在NNS终止中的作用，我们对总RNA进行了RNA测序（RNA-Seq），并对RNA聚合酶II（RNAPII）定位进行了ChIP-exo分析。RRP 6的缺失促进了多种NNS依赖性转录物的过度延伸，这是由不正确加工的3' RNA末端和特定靶基因处的错误转录物终止引起的。RNAPII终止的缺陷通过转录干扰和/或反义抑制引起mRNA表达的转录组范围的变化，类似于先前报道的从细胞核中耗尽Nrd 1的影响。在所有已知的NNS靶标中鉴定出延长的转录物，其中转录终止的最大变化发生在CUT。有趣的是，我们在我们的研究中检测到的延伸转录物在许多位置显示出与Nrd 1未终止转录物的显著相似性，这表明Rrp 6除了在特定靶点的3'端RNA加工和/或降解外，还与NNS复合物整体地促进转录终止。RNAPII负责蛋白质编码基因和短的调节RNA的转录。在酿酒酵母中，RNAPII转录的RNA的终止≤1000个碱基需要NNS复合物（由Nrd 1，Nab 3和Sen 1组成），由外泌体加工，以及核特异性催化亚基Rrp 6。研究表明，Rrp 6与Nrd 1直接相互作用，但Rrp 6是否是NNS依赖性终止所必需的尚不清楚。Rrp 6功能的丧失可能导致NNS依赖性转录物的延伸（或终止的抑制），或者Rrp 6可能仅在进行RNA 3'末端加工的事实之后起作用。在这里，我们进行了深入的差异表达分析，并比较RNA测序数据的转录长度和丰度在细胞缺乏RRP 6的RNAPII定位的ChIP-exo分析。我们发现许多转录本在rrp 6 Δ中具有相似的长度，并且下游基因的表达水平显著降低，这些转录本在Nrd 1活性丧失后被定义为未终止的。这表明在缺乏Nrd 1或Rrp 6的细胞中发生了类似的转录干扰机制。事实上，我们发现增加RNAPII位于其终止位点的下游，在许多已知的Nrd 1调节转录。总体而言，我们的研究结果清楚地表明，Rrp 6活性是需要有效的NNS终止在体内。
The exosome and its nuclear specific subunit Rrp6 form a 3’-5’ exonuclease complex that regulates diverse aspects of RNA biology including 3’ end processing and degradation of a variety of noncoding RNAs (ncRNAs) and unstable transcripts. Known targets of the nuclear exosome include short (<1000 bp) RNAPII transcripts such as small noncoding RNAs (snRNAs), cryptic unstable transcripts (CUTs), and some stable unannotated transcripts (SUTs) that are terminated by an Nrd1, Nab3, and Sen1 (NNS) dependent mechanism. NNS-dependent termination is coupled to RNA 3’ end processing and/or degradation by the Rrp6/exosome in yeast. Recent work suggests Nrd1 is necessary for transcriptome surveillance, regulating promoter directionality and suppressing antisense transcription independently of, or prior to, Rrp6 activity. It remains unclear whether Rrp6 is directly involved in termination; however, Rrp6 has been implicated in the 3’ end processing and degradation of ncRNA transcripts including CUTs. To determine the role of Rrp6 in NNS termination globally, we performed RNA sequencing (RNA-Seq) on total RNA and perform ChIP-exo analysis of RNA Polymerase II (RNAPII) localization. Deletion of RRP6 promotes hyper-elongation of multiple NNS-dependent transcripts resulting from both improperly processed 3’ RNA ends and faulty transcript termination at specific target genes. The defects in RNAPII termination cause transcriptome-wide changes in mRNA expression through transcription interference and/or antisense repression, similar to previously reported effects of depleting Nrd1 from the nucleus. Elongated transcripts were identified within all classes of known NNS targets with the largest changes in transcription termination occurring at CUTs. Interestingly, the extended transcripts that we have detected in our studies show remarkable similarity to Nrd1-unterminated transcripts at many locations, suggesting that Rrp6 acts with the NNS complex globally to promote transcription termination in addition to 3’ end RNA processing and/or degradation at specific targets. RNAPII is responsible for transcription of protein-coding genes and short, regulatory RNAs. In Saccharomyces cerevisiae, termination of RNAPII-transcribed RNAs ≤1000 bases requires the NNS complex (comprised of Nrd1, Nab3, and Sen1), processing by the exosome, and the nuclear specific catalytic subunit, Rrp6. It has been shown that Rrp6 interacts directly with Nrd1, but whether or not Rrp6 is required for NNS-dependent termination is unclear. Loss of Rrp6 function may result in extension (or inhibition of termination) of NNS-dependent transcripts, or Rrp6 may only function after the fact to carry out RNA 3’ end processing. Here, we performed in-depth differential expression analyses and compare RNA-sequencing data of transcript length and abundance in cells lacking RRP6 to ChIP-exo analysis of RNAPII localization. We find many transcripts that were defined as unterminated upon loss of Nrd1 activity are of similar length in rrp6Δ, and expression levels of downstream genes are significantly decreased. This suggests a similar transcription interference mechanism occurs in cells lacking either Nrd1 or Rrp6. Indeed we find increased RNAPII located downstream of its termination site at many know Nrd1-regulated transcripts. Overall, our findings clearly demonstrate that Rrp6 activity is required for efficient NNS termination in vivo.
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