Post-transcriptional monoadenylation by TENT2 terminates human RNA polymerase III transcript 3' end processing and promotes 7SL RNA biogenesis [gene_specific_RNAseq]

Small non-coding RNAs (sncRNAs) are subject to a variety of 3' end trimming and tailing activities during biogenesis, which dictate whether they undergo maturation or are instead subjected to degradation. To investigate the dynamics of human sncRNA 3' end processing at a global level we performed genome-wide 3' end sequencing of nascently-transcribed and steady state sncRNAs. This revealed widespread post-transcriptional adenylation of nascent sncRNAs, which came in two distinct varieties. One is characterized by oligoadenylation, which is transient, promoted by TENT4A/4B polymerases, and observed primarily on unstable sno- and scaRNAs that are not fully processed at their 3' ends. The other is characterized by monoadenylation, which is broadly catalyzed by TENT2 and stably accumulates at the 3’-end of many RNA Polymerase-III-transcribed (Pol-III) RNAs as well as a subset of small nuclear RNAs. This monoadenylation event terminates 3' end uridylation/deuridylation dynamics characteristic of nascent Pol-III RNAs and, in the case of 7SL RNAs, prevents their accumulation with nuclear La protein and promotes their biogenesis towards assembly into cytoplasmic signal recognition particles. RNAs from control or TENT2-depleted conditions were gene-specifically PCR amplified for 3' end sequencing. RNAs associated with La and SRP54 during IP were PCR amplified for 3' end sequencing. RNAs labeled with s4U (two 3-hour pulses) in control or TENT2 KO conditions were PCR amplified for 3' end sequencing.

小非编码RNA（small non-coding RNAs，sncRNAs）在生物发生过程中会经历多种3'端修剪与尾端修饰活动，这一过程决定了其是走向成熟还是被降解。为在全局层面解析人类sncRNA 3'端加工的动态变化，我们对新生转录及稳态的sncRNA开展了全基因组3'端测序。该研究揭示了新生sncRNA广泛存在的转录后腺苷酸化现象，且该现象存在两种截然不同的类型。其一为寡腺苷酸化型，该过程具有瞬时性，由TENT4A/4B聚合酶介导，主要见于3'端未完全加工的不稳定小核仁RNA（snoRNAs）及小卡哈尔体特异性RNA（scaRNAs）。其二为单腺苷酸化型，该过程广泛由TENT2催化，并在众多RNA聚合酶III（RNA Polymerase-III，Pol-III）转录的RNA以及部分小核RNA的3'端稳定积累。这种单腺苷酸化事件会终止新生Pol-III RNA所特有的3'端尿苷酸化/去尿苷酸化动态过程；以7SL RNA为例，该修饰可阻止其与核La蛋白结合，并促进其生物发生过程，最终组装为细胞质信号识别颗粒。针对对照组或TENT2敲低条件下的RNA，我们通过基因特异性PCR扩增后开展3'端测序；免疫沉淀（immunoprecipitation，IP）过程中与La蛋白及SRP54结合的RNA，经PCR扩增后开展3'端测序；对照组或TENT2敲除（knockout，KO）条件下经4-硫尿苷（s4U）标记（两次3小时脉冲标记）的RNA，经PCR扩增后开展3'端测序。