Regulatory functions of nascent RNA

Nascent pre-mRNA is usually considered to be a passive intermediary that lacks a regulatory role. However, results from our lab and others indicate that pre-mRNA plays a regulatory role in the cell.

It is well established that transcription of DNA into coding and non-coding (nc)RNAs is regulated by proteins that respond to DNA sequence composition and chromatin state. It is also becoming clear that RNA molecules can themselves play a role in transcriptional and chromatin regulation. Specific long non-coding RNA (lncRNA) species have been found to interact with chromatin regulatory proteins and promote or inhibit their recruitment to sites on chromatin. In contrast, nascent pre-mRNA is primarily considered to be a passive intermediary that lacks a regulatory role.

Regulation of PRC2

Results from our lab and others indicate that pre-mRNA also plays a regulatory role in the cell. Using RNA IP, we discovered that the repressive chromatin modifier PRC2 interacts with RNAs transcribed from protein coding genes (Kanhere et al., 2010). Using iCLIP, that identifies directly-occurring protein-RNA interactions, we determined that these PRC2-bound transcripts were pre-mRNAs (Beltran et al, 2016). Within pre-mRNAs, PRC2 preferentially interacts with G-tract sequences located in the first intron that have the capacity to fold into G-quadruplex structures (Beltran et al, 2019). G4 RNA blocks the interaction of PRC2 with nucleosomes and inhibits its catalytic activity in vitro (Beltran et al, 2016; Beltran et al., 2019). In cells, inhibiting Pol II or degrading RNA induces recruitment of PRC2 to chromatin at active genes (Beltran et al., 2016). Furthermore, we demonstrated that tethering G4 RNA to PRC2 target genes using dCas9 is sufficient to evict PRC2 from chromatin and erase the repressive state (Beltran et al., 2019). These data support the hypothesis that nascent RNA inhibits recruitment of PRC2 to prevent inappropriate silencing of active genes (Fig. 1).

Figure 1. A model for the relationship between PRC2 RNA and chromatin binding.
<h4>Figure 1. A model for the relationship between PRC2 RNA and chromatin binding.</h4><p> PRC2 can bind either to chromatin or to RNA in a mutually antagonistic fashion (centre). At lowly expressed genes, there is active competition between chromatin and RNA for PRC2 binding. At highly active genes (right), repeated rounds of nascent RNA synthesis outcompete chromatin for PRC2 binding, protecting genes from inappropriate silencing. Figure taken from Beltran et al., 2016.</p>

Regulation of other proteins

We have recently sought to determine whether the repressive effect of pre-mRNA might extend to other regulatory proteins. We have used proteomics methods to identify the changes in chromatin composition caused by transcriptional inhibition or RNA degradation (Skalska et al., 2021). Strikingly, we found that nascent RNA inhibited the interaction of a set of proteins significantly enriched for functions in pluripotency, chromatin organisation and gene expression. For some of these factors, RNA inhibited their interaction with nucleosomes, as is also the case for PRC2. For the transcriptional elongation factor P-TEFb, nascent RNA inhibited interaction with chromatin in a manner regulated by the 7SK ribonucleoprotein complex. These data support a model in which nascent RNA has a central role in providing direct feedback from transcriptional output to chromatin state (Skalska et al., 2017).

Figure 2. Model for the effect of nascent RNA on the interaction of regulatory proteins with chromatin.
Figure 2. Model for the effect of nascent RNA on the interaction of regulatory proteins with chromatin.Pre-mRNA and other nascent transcripts bind a set of transcriptional and chromatin regulators and antagonise their association with chromatin. For some of these factors, RNA inhibits their interaction with nucleosomes. For P-TEFb, RNA binding and recruitment to chromatin are regulated by the 7SK RNP. Higher order chromatin structure may concentrate the antagonistic effects of RNA at regions of active chromatin in the cell. Figure taken from Skalska et al., 2021.