EXOonRNA · Delineating the kinetic RNA interactome of nuclear exosome adaptor complexes

Delineating the kinetic RNA interactome of nuclear exosome adaptor complexes

The human genome is transcribed extensively, but most transcription events do not result in functional RNA molecules. Therefore, much of the RNA output of the genome is regulated at the level of RNA degradation, a key gene regulatory mechanism that remains significantly understudied. Cells must distinguish functional RNAs from non-functional ones and efficiently eliminate excess transcripts, which would otherwise interfere with other RNA-related processes. The RNA exosome complex is the primary molecular machine responsible for degrading newly produced RNA in eukaryotic nuclei (Figure 1a). It filters out RNAs marked for degradation from their functional counterparts with the help of adapter complexes, specifically the nuclear exosome targeting (NEXT) complex and the polyA-tail exosome targeting (PAXT) connection. Both of these adapter complexes consist of several RNA-binding proteins (RBPs) that interact with RNA, but exactly how they distinguish functional from non-functional RNA remains a mystery in molecular biology. This makes it crucial to uncover the principles by which nuclear exosome adaptors target RNA. This need is emphasized by findings that abnormal RNA levels are strongly linked to human diseases and that both nuclear RNA degradation and ExoAC complexes are connected to disease biology. Therefore, sorting nuclear RNA for degradation is a fundamental challenge in gene regulation with important implications for biomedicine. Moreover, with the rise of RNA-based technologies, such as mRNA vaccines, a deep understanding of RNA decay pathways is essential for future advancements in these areas. In the proposed project, I aimed to identify critical checkpoints in nuclear RNA biogenesis that help distinguish functional from non-functional RNA. To achieve this, I have studied the RNA-binding components of the NEXT and PAXT ExoACs, namely RBM7 and LENG8 proteins, using a crosslinking followed by immunoprecipitation (CLIP) method (Objective 1). I hypothesize that to promote RNA decay via the nuclear exosome, the RNA-binding components of ExoACs assemble on RNA substrates in a stepwise manner, ultimately marking the substrate for degradation. To test this, I analyzed RNA-binding maps of key proteins associated with the nuclear exosome adapter complexes (Objective 2). On the basis of above research work I concluded the key principles how two different ExoAC complexes NEXT and PAXT target RNAs in the nucleus (Figure 1b). Namely, I found that NEXT interacts with short non-polyadenylated RNAs co-transcriptionally, just after their synthesis. Conversely, PAXT approaches polyadenylated RNAs at the late stages of their biogenesis in the nucleus, where it competes with export machinery for RNAs as parts of ribonucleoprotein particles (RNPs) with shared features with export-competent RNPs. This results shed the light on the features of RNA but also interacting RBPs, associated either with nuclear decay or export to the cytoplasm.

Data: CORDIS, © European Union