Surveillance-ready transcription: nuclear RNA decay as a default fate

Abstract

Eukaryotic cells synthesize enormous quantities of RNA from diverse classes, most of which are subject to extensive processing. These processes are inherently error-prone, and cells have evolved robust quality control mechanisms to selectively remove aberrant transcripts. These surveillance pathways monitor all aspects of nuclear RNA biogenesis, and in addition remove nonfunctional transcripts arising from spurious transcription and a host of non-protein-coding RNAs (ncRNAs). Surprisingly, this is largely accomplished with only a handful of RNA decay enzymes. It has, therefore, been unclear how these factors efficiently distinguish between functional RNAs and huge numbers of diverse transcripts that must be degraded. Here we describe how bona fide transcripts are specifically protected, particularly by 5' and 3' modifications. Conversely, a plethora of factors associated with the nascent transcripts all act to recruit the RNA quality control, surveillance and degradation machinery. We conclude that initiating RNAPII is 'surveillance ready', with degradation being a default fate for all transcripts that lack specific protective features. We further postulate that this promiscuity is a key feature that allowed the proliferation of vast numbers of ncRNAs in eukaryotes, including humans.

Keywords: RNA processing; RNA surveillance; gene expression; quality control.

Figure 1.

Processing and surveillance of pre-mRNAs. Multiple steps during mRNA transcription and processing are screened by surveillance activities. (a) Delayed or aberrant capping leads to decay by nuclear 5′ surveillance pathways. Degradation requires a pyrophosphatase activity (orange circle) to remove the triphosphate and a coupled 5′–3′ exonuclease (orange pacman). Correctly maturing transcripts are protected by the presence of the m⁷G cap and the cap binding complex (CBC; grey triangle). Following normal transcript cleavage and polyadenylation, the 3′ fragment of the nascent transcript is targeted by the 5′ exonuclease in order to terminate RNAPII transcription. (b) Prematurely terminated transcripts are 3′ degraded by the nuclear exosome (blue pacman). Transcription termination and surveillance can involve either complete dissociation of the polymerase (left) or polymerase backtracking to reveal the 3′ end, providing an entry point for the exosome (right). (c) Unspliced transcripts are targeted by the surveillance machinery. In normal mRNA biogenesis, introns are typically spliced cotranscriptionally. Excised introns must be constitutively degraded and features associated with splicing or introns may act to recruit the nuclear surveillance machinery. When introns are not efficiently removed, these factors may facilitate degradation of the entire transcript. (d) Aberrant 3′ end formation leads to surveillance by the nuclear exosome. In fission yeast, this can involve RNAPII stalling and backtracking downstream of the PAS (centre). Alternatively, the budding yeast protein Reb1 (red circle) can terminate transcription by functioning as a roadblock (right). RNAPII is ubiquitinated and degraded, and the released transcript is degraded by the nuclear exosome. Correctly terminated transcripts (left) are protected by a poly(A) tail appropriately packaged with poly(A) binding proteins (green circles). (e) Transcripts with prolonged nuclear retention are subject to slow, default surveillance pathways. This process appears to be facilitated in part by nuclear poly(A) binding proteins, which protect the transcript but can also stimulate decay through recruitment of the nuclear surveillance machinery.

Figure 2.

Protective features in RNA stability. (a) Primary RNAPII transcripts are initially protected by the terminal 5′ triphosphate, which blocks degradation by the nuclear 5′–3′ exonucleases Rat1/Xrn2. Transcripts are generally rapidly modified by addition of an inverted GpppN cap structure. This is sensitive to removal by the pyrophosphatases Rai1 and DXO, but undergoes m⁷G methylation and association with the cap binding complex (CBC), conferring pyrophosphatase resistance. (b) Most mRNAs are shielded at their 3′ end by a poly(A) tail packaged with poly(A) binding proteins. The non-polyadenylated, replication-dependent histone mRNAs are protected by a terminal stem–loop structure bound to the stem–loop binding protein (SLBP). (c) Small nucleolar RNAs (snoRNAs) and small nuclear RNAs (snRNAs) are shielded from exosome-mediated decay by specific proteins bound to the 3′ end. snRNAs and many snoRNAs are protected at their 5′ end by the trimethylated m^2,2,7G cap. (d) The mature MALAT1 transcript contains a triple helix that sequesters the 3′ end and prevents 3′–5′ exonucleolytic decay.

Figure 3.

Pro-surveillance factors in RNAPII transcription units. Nascent transcripts contain numerous features which facilitate the recruitment of surveillance factors. Importantly, these factors are also present during normal RNA biogenesis, which presumably allows the nuclear surveillance machinery to ‘inspect’ all nascent RNAs. Details are discussed in the text. h, human; Sc, Saccharomyces cerevisiae.

Figure 4.

Major classes of noncoding RNA. The regions surrounding eukaryotic protein-coding genes generate a set of ncRNAs in addition to the mRNA transcript. These include: (1) short divergent promoter associated transcripts from the nucleosome-free promoter region, (2) antisense (as) transcripts from the nucleosome-depleted terminator region, (3) enhancer-associated eRNAs. In addition, (4) a range of ncRNAs are transcribed from intergenic locations. h, human; Sc, Saccharomyces cerevisiae.