RNA surveillance and the initial steps of RNA biogenesis

Abstract

The human genome is pervasively transcribed by RNA polymerases, yet only a small portion encodes proteins. Many transcripts are non-coding RNAs (ncRNAs), including cryptic unstable transcripts (CUTs), which are rapidly degraded by nuclear RNA surveillance. In Schizosaccharomyces pombe, the conserved 11-subunit MTREC complex targets CUTs to the nuclear exosome for degradation, but how MTREC distinguishes CUTs from mRNAs remains unclear. A key submodule is the conserved CBCA complex, consisting of the cap-binding proteins Cbc1 and Cbc2, and Arsenite-resistance protein 2 (Ars2). CBCA binds the 5' cap of nearly all RNAs. Ars2 serves as the bridge between CBCA and the scaffold protein Red1, potentially linking all capped RNAs to MTREC. Properly processed mature mRNAs escape MTREC-mediated degradation, but the mechanism underlying this selectivity is not understood. Our aim was to investigate how Ars2 contributes to this process. To test the importance of Red1-Ars2 interaction, we introduced point mutations and short deletions in Red1 that disrupt its association with Ars2, impairing MTREC recruitment. Biochemical assays confirmed the loss of interaction between Ars2 and Red1, while RNA-seq revealed that under nitrogen starvation, but not normal growth conditions, short CUTs accumulate in these mutants. This indicates that the intact Ars2-CBCA module is required for efficient recognition and degradation of CUTs under stress. The phenotypes, however, were mild, suggesting redundancy with other nuclear exosome cofactors, potentially analogous to the mammalian NEXT complex. Additional biochemical data further suggest a length-dependent model of substrate selection. Short RNAs are targeted to the MTREC/exosome pathway via the CBCA subcomplex, unless protected by transcript-specific rescue factors such as FLASH (histone mRNAs) or PHAX (snRNAs). In contrast, longer RNAs, such as mRNAs, appear to evade degradation because when the RNA export factor Mlo3 is recruited to the CBC complex, it cannot coexist with Ars2. We therefore propose that Ars2 initially associates broadly with all nascent RNA Pol II transcripts, but transcript fate diverges according to length: short RNAs are retained and degraded by Ars2-MTREC mediated exosome degradation, whereas long RNAs are saved through Mlo3-mediated displacement of Ars2 and subsequent RNA export. The MTREC complex also plays an important role, in coordination with the CNM (Ctr1-Nrl1-Mtl1) and WGG (Wdr83-Gpl1-Gih35) complexes, in recognising and degrading mRNAs that activate quality control mechanisms during splicing. Through machine learning, RNA-seq, RNA-IP, structural analyses, and mass spectrometry, we show that CNM and WGG recognise stalled spliceosomes containing pre-mRNAs with suboptimal 5' splice sites. We further reveal that WGG maintains these spliceosomes in a catalytically dormant state and recruits CNM and disassembly factors to form a spliceosome intermediate primed for discard, termed the Bd complex. Our results also demonstrate that CNM facilitates transfer of these aberrant transcripts to MTREC for exosomal degradation, with Ctr1-Prp43 interactions contributing to spliceosome disassembly. Together, these results uncover a coordinated surveillance network that selectively eliminates defective RNAs, highlighting a multi-layered strategy for preserving transcriptome fidelity. Defects in such pathways could contribute to the accumulation of aberrant RNAs in diseases such as cancer or neurodegeneration, highlighting the broader relevance of RNA quality control mechanisms. Show more