Alternative RNA Stuctures Formed During Transcription Depend on Elongation Rate and Modify RNA processing

Most RNA processing occurs co-transcriptionally. We interrogated nascent pol II transcripts by chemical and enzymatic probing, and determined how the “nascent RNA structureome” relates to splicing, A-I editing and transcription speed. RNA folding within introns and steep structural transitions at splice sites are associated with efficient co-transcriptional splicing. A slow pol II mutant elicits extensive remodeling into more folded conformations with increased A-I editing. Introns that become more structured at their 3' splice sites get co-transcriptionally excised more efficiently. Slow pol II altered folding of intronic Alu elements where cryptic splicing and intron retention are stimulated, an outcome mimicked by UV which decelerates transcription. Slow transcription also remodeled RNA folding around alternative exons in distinct ways that predict whether skipping or inclusion is favored, even though it occurs post-transcriptionally. Hence co-transcriptional RNA folding modulates post-transcriptional alternative splicing. In summary the plasticity of nascent transcripts has widespread effects on RNA processing. Overall design: RNA pol II immunoprecipiation in isogenic HEK293 cells expressing a slow pol II mutant or wild type pol II was combinded with either in vivo chemical RNA structure probing using DMS or enzymatic probing using structure specific nuleases to identify cotranscriptional RNA structures

绝大多数RNA加工事件均以共转录（co-transcriptional）方式进行。我们借助化学探针与酶促探针技术，对新生RNA聚合酶II（RNA polymerase II, Pol II）转录本开展系统性表征，解析了"新生RNA结构组（nascent RNA structureome）"与剪接、A-I RNA编辑（A-I editing）及转录速率之间的关联。
研究发现，内含子区域内的RNA折叠行为以及剪接位点处的剧烈结构转变，与高效的共转录剪接过程密切相关。携带慢转录速率突变的Pol II突变体可引发广泛的构象重塑，使转录本形成更为稳定的折叠构象，同时伴随A-I RNA编辑水平的提升。
在3'剪接位点处折叠程度更高的内含子，其共转录剪切除过程更为高效。慢转录速率的Pol II突变体改变了内含子Alu元件的折叠状态，该区域的隐蔽剪接与内含子滞留现象均被激活；而通过紫外线（UV）减速转录过程也可重现这一实验结果。
慢转录过程还通过独特的方式重塑了可变外显子周边区域的RNA折叠状态，这种重塑特征可用于预测可变剪接中更倾向于发生外显子跳跃还是外显子保留，尽管该可变剪接事件本身属于转录后过程。因此，共转录阶段的RNA折叠可调控转录后的可变剪接过程。
综上，新生转录本的构象可塑性对RNA加工过程具有广泛的调控作用。
总实验设计：在表达慢突变型Pol II或野生型Pol II的同基因HEK293细胞系中开展RNA Pol II免疫沉淀实验，并将其与两种检测方法相结合：一是采用硫酸二甲酯（dimethyl sulfate, DMS）进行体内化学RNA结构探针检测，二是采用结构特异性核酸酶（structure-specific nucleases）开展酶促探针检测，以此实现共转录RNA结构的鉴定。