Periodic Reporting for period 1 - RNAfate (Identifying RNA fate checkpoints by resolving the high-resolution spatiotemporal binding dynamics of CBC containing complexes)
Berichtszeitraum: 2019-04-01 bis 2021-03-31
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?
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?
The ‘gold standard’ method to study in vivo protein-RNA interactions is by UV cross-linking and immunoprecipitation (CLIP) coupled with high-throughput RNA sequencing. UV irradiation covalently links protein-RNA complexes, enabling robust recovery of interacting RNAs by immunoprecipitation (IP). Recovered RNAs are processed into libraries for high-throughput sequencing. Subsequent analysis of CLIP libraries can yield information regarding the positional distribution of the RBP throughout the transcriptome at nucleotide resolution. In order to address the objectives outlined above, we were required to capture early RNP dynamics, however, current CLIP protocols do not provide the resolution necessary as they only capture steady state protein-RNA interactions. For this purpose, we developed temporal individual resolution CLIP (tiCLIP), which combines transient transcriptional synchronisation of RNAPII followed by UV cross-linking and immunoprecipitation of samples at serial timepoints (Fig1B).
I implemented the tiCLIP protocol on ALYREF, PHAX, CBP20, CBP80 and RBM7. After leveraging the tiCLIP positional data across all transcription units (genes) I found that the CBC-cofactors (ALYREF, RBM7 and PHAX) all displayed similar co-transcriptional RNA binding profiles. Importantly, these RNA binding profiles are common to all RNAPII transcripts and progress temporally through genes at the speed of transcription, indicating that the CBC-cofactors rapidly sample the emerging transcript. Thereafter, stable association to defined RNA elements or gene structures is triggered by RNA metabolic events, such as splicing. More specifically, I utilised the first and second transesterfication reactions that occur during splicing as a molecular yard stick, and could temporally separate the stable association of ALYREF and RBM7 with nascent transcripts. Unexpectedly from these analyses, I found that the CBC component CBP80 whilst found enriched at the 5’ end of transcripts, could also be found downstream inside transcription units and behaved more like the CBC-cofactors sampling the elongating nascent transcript, which indicates that the protein components of the CBC may not associate with RNA in a strictly similar manner.
These data have been presented at 6 international conferences including 2 oral presentation. Both the research findings and methodology are currently being prepared as separate manuscripts for submission at peer-reviewed journals.
I implemented the tiCLIP protocol on ALYREF, PHAX, CBP20, CBP80 and RBM7. After leveraging the tiCLIP positional data across all transcription units (genes) I found that the CBC-cofactors (ALYREF, RBM7 and PHAX) all displayed similar co-transcriptional RNA binding profiles. Importantly, these RNA binding profiles are common to all RNAPII transcripts and progress temporally through genes at the speed of transcription, indicating that the CBC-cofactors rapidly sample the emerging transcript. Thereafter, stable association to defined RNA elements or gene structures is triggered by RNA metabolic events, such as splicing. More specifically, I utilised the first and second transesterfication reactions that occur during splicing as a molecular yard stick, and could temporally separate the stable association of ALYREF and RBM7 with nascent transcripts. Unexpectedly from these analyses, I found that the CBC component CBP80 whilst found enriched at the 5’ end of transcripts, could also be found downstream inside transcription units and behaved more like the CBC-cofactors sampling the elongating nascent transcript, which indicates that the protein components of the CBC may not associate with RNA in a strictly similar manner.
These data have been presented at 6 international conferences including 2 oral presentation. Both the research findings and methodology are currently being prepared as separate manuscripts for submission at peer-reviewed journals.
I have captured the co-transcriptional RNA binding profiles of selected factors at a temporal resolution not previously achieved. Critical check points undertaken by the cell to determine the fate of an RNA happen during the early phases of transcription. The novel tiCLIP approach described here enables assessment of these early steps. In addition, tiCLIP could also easily be applied more generally to understand how dynamic protein-RNA interaction networks are rapidly rewired in response to a genotoxic/cellular stress or molecular event.