The human genome is pervasively transcribed. High-throughput transcriptome analyses across 15 human cell lines have identified the surprising fact that more than 80% of DNA is biochemically active and undergoes at least one transcription event. A major subset of these are transcribed by RNA polymerase II (RNAPII), such as long noncoding RNAs (lncRNAs). Most of these molecules are extremely labile and hereby differ from the better described stable ncRNAs (rRNAs, tRNAs, snRNAs and snoRNAs), and small regulatory RNAs (miRNAs, siRNAs, piRNAs). Being transcribed by RNAPII, lncRNAs share common features with mRNAs, such as 5’ m7-G caps, however, the general fates and functions of these transcripts differ vastly. That is, the majority of correctly processed mRNAs are exported to the cytoplasm to participate in protein synthesis; whereas, a large fraction of lncRNAs is nuclear retained and often rapidly degraded.
Given these varied characteristics of mRNAs and lncRNAs, the overarching aim of this project is to decipher how the cell sorts these different RNAs into their appropriate processing/export or turnover pathways and when remodelling of the early ribonucleoprotein particle (RNP) dictates RNA fate? Understanding this problem is of immense importance in modern molecular biology as it will help explain how cells cope with such massive genomic output without erroneously degrading functional RNAs or leaving spurious transcripts unrecognised by cellular quality control.
RNA fate is ultimately dictated by the proteins with which the nascent transcript associates. The 5’ m7-G cap of the 20-40 nt long nascent RNA is a hallmark of RNAPII-derived transcripts. Through its association with the 5’ cap, the cap-binding complex (CBC), composed of the CBP20 and CBP80 proteins, serves as a landing pad for factors to associate with the elongating nascent RNA; forming the early and growing ribonucleoprotein (RNP) complex (Fig. 1A). The CBC is essential for a multitude of nuclear RNA metabolic events, such as turnover and transport. In order to coordinate these vital processes, the CBC connects to various RNA export and decay factors to elicit productive or destructive metabolic functions, respectively. For example, the transcription termination and turnover of PROMPTs, eRNAs and other early terminated transcripts are also mediated by the CBC. RNA turnover is induced by the CBC recruiting the ZC3H18 Zn-finger protein and the Nuclear EXosome Targeting (NEXT) complex (composed of RBM7, MTR4 and ZCCHC8) forming the so-called CBC-NEXT (CBCN) assembly (Fig. 1), which promotes RNA degradation by the ribonucleolytic exosome complex. Nuclear export of sn(o)RNAs is mediated by the CBC-interacting PHosphorylated Adapter RNA eXport (PHAX) protein, forming the CBCAP complex (Fig. 1A). Finally, export of mRNAs is mediated through the CBC interacting with the adaptor proteins ALYREF (Fig. 1A), which is essential components of the human transcription export complex, hTREX.
The overarching aim of this project is to decipher how the cell sorts different RNAs into their appropriate metabolic pathways, by delineating which nascent RNP features dictate RNA fate. Since the CBC and its interaction partners partake in defining the early RNP, and functions as a landing pad to recruit both productive (PHAX & ALY/REF) and destructive (NEXT) cofactors (Fig. 1A), it provides an excellent dichotomic system to study this question. I therefore hypothesise that a description of proteins recruited to the early and emerging RNP will allow me to answer key questions about which factors and RNA/DNA elements establish the sorting mechanisms that cells employ to decide RNA fate. In order to do so two specific aims will be pursued:
1) Deciphering the spatiotemporal recruitment of the CBC and its cofactors during early RNP formation - when and where are the different proteins loaded?
2) Identifying the RNA/DNA elements contributing to RNA fate decisions – what are the RNA fate checkpoints?
