The multitasking polyA tail: nuclear RNA maturation, degradation and export

Abstract

A polyA (pA) tail is an essential modification added to the 3' ends of a wide range of RNAs at different stages of their metabolism. Here, we describe the main sources of polyadenylation and outline their underlying biochemical interactions within the nuclei of budding yeast Saccharomyces cerevisiae, human cells and, when relevant, the fission yeast Schizosaccharomyces pombe Polyadenylation mediated by the S. cerevisiae Trf4/5 enzymes, and their human homologues PAPD5/7, typically leads to the 3'-end trimming or complete decay of non-coding RNAs. By contrast, the primary function of canonical pA polymerases (PAPs) is to produce stable and nuclear export-competent mRNAs. However, this dichotomy is becoming increasingly blurred, at least in S. pombe and human cells, where polyadenylation mediated by canonical PAPs may also result in transcript decay.This article is part of the theme issue '5' and 3' modifications controlling RNA degradation'.

Keywords: RNA decay; RNA export; RNA polyadenylation; TRAMP complex; polyA binding proteins; transcription termination.

Figure 1.

Model of CPF/CPSF-mediated mRNA 3'-end formation and nuclear export. (a) Schematic overview of 3′-end cleavage, polyadenylation and export factors from S. cerevisiae (a(i)) and human (a(ii)) cells. Factors with no human or S. cerevsiae orthologues with a similar function in polyadenylation and export, respectively, are marked with dashed outlines. In some cases, the human orthologue has not yet been studied in the context of cleavage and polyadenylation. The CPF and CPSF complexes are composed of three basic modules (inspired from model in [20]): (i) the endonuclease module organized around the Ysh1/CPSF73 enzyme supported by Cft2/CPSF100 and Mpe1; (ii) the RNAPII phosphatase module, the catalytic activity of which is mediated by the Glc7/PP1 and Ssu72 subunits of the ‘associated with Pta1′ (human symplekin protein – SYM*) complex (APT: Pta1, Swd2, Ref2, Pti1, Syc1); and (iii) the pA polymerase module, containing the Pap1/PAPOLA/PAPOLG/Star-PAP enzymes, which are recruited to this module mainly by an interaction with Cft1/CPSF160 but also with Fip1/FIP1, Pfs2/WDR33 and Yth1/CPSF30. The yeast CPF complex is further assisted by Cleavage Factor I (CFI: Clp1, Hrp1, Pcf11, Rna14, Rna15), while the human CPSF complex is supported by the CFI-like sub-complexes: cleavage stimulation factor (CstF: CstF50/77/64), cleavage factor II (CFIIm: CLP1, PCF11) and the human-specific cleavage factor I (CFIm: CFIm25/59/68). The CPF and CPSF complexes are recruited dually by binding to specific sequences in the nascent RNA and to the RNAPII CTD. In S. cerevisiae cells, polyadenylation is shown to be stimulated by the THO complex (indicated by the arrow) and in human cells by PABPN1 (arrow). The formation of an export-competent mRNP starts co-transcriptionally with association of the cap-binding complex (CBC, CBP20/80). In yeast Npl3, TREX and Nab2 loading enhances recruitment of the export adaptor Mex67/Mtr2, while in human only the TREX subunit Aly/REF stimulates nuclear export. Successful synthesis of a long pA tail and mRNP assembly leads to release of the newly made RNA from the site of transcription and its translocation to the nuclear pore. Mex67/NXF1 interaction with core FG-nuclear pore factors and Nab2 interactions with the Gfd1 NPC subunit stimulates export. Nab2 binding to Mlp1/Mlp2 promotes retention of unspliced transcripts. An mRNP associated helicase Dbp5/DDX19B is activated by interaction with the Gle1/GLE1 NPC subunit located at the cytoplasmic face and contributes to the displacement of Nab2 and possibly other nuclear mRNP subunits from the transcript. Other mechanisms also contribute to recycling of export factors, such as phosphorylation in case of Npl3. This allows for the release of the transcript into the cytoplasm. Human PABPN1 shuttles between the nucleus and the cytoplasm, but the mechanism by which it is exchanged by cytoplasmic PABPC1 is still unclear. (b) Schematic overview of domain and motif organizations of the main CPF-associated non-orthologous PABs in S. cerevisiae (b(i) Nab2) and human (b(ii) PABPN1) cells highlights the diverse functions of these proteins in pA tail-length control and RNA nuclear export. PABPN1 binds RNA using an RNA Recognition Motif (RRM), while Nab2 recognizes RNA via its three distal zinc fingers (ZnFs) [21]. PABPN1 interacts directly with and stimulates PAPOLA activity, while Nab2 binds the NPC subunits Mlp1/2 and Gfd1 through its N-terminal domain. Moreover, PABPN1 interacts with the spliceosome subunit SKIP [22]. The region responsible for nuclear import of Nab2 is located within the RGG domain [23].

Figure 2.

Nuclear RNA decay pathways in S. pombe and human cells. (a) Schematic overview of the different modules of the S. pombe MTREC/NURS complex. Mtl1 and Red1 are central proteins that are suggested to facilitate the recruitment of the various modules to the RNA exosome. The Mmi1-Iss10 module, assisted by Pab2, plays a key role in the removal of meiotic mRNAs during vegetative growth. The modules binding the RNA 5′ cap (Cbc1-Cbc2-Ars2) and 3′-end pA tail (Pab2-Rmn1-Red5) mediate the removal of CUTs. Finally, the Mtl1–Ctr1–Nrl1 complex interacts with the spliceosome and is involved in the recognition and degradation of unspliced or mis-spliced RNA. Also depicted is the S. pombe TRAMP complex, composed of Cid14, Air1 and the Mtl1 paralog Mtr4. This complex targets nucleolar tRNAs and rRNAs for decay. (b) Schematic overview of nucleoplasmic human complexes involved in RNA targeting. The cap-binding complex (CBC), composed of CBC20 and CBC80, is bridged via the ARS2 and ZC3H18 proteins to the NEXT complex composed of the RNA-binding protein RBM7, the Zn-finger protein ZCCHC8 and MTR4, forming the CBC-NEXT (CBCN) complex [69,70]. The NEXT complex, which does not appear to have a S. pombe orthologous module, targets short cryptic RNAs, like PROMPTs and enhancer RNAs (eRNAs), for exosomal decay. PABPN1, on the other hand, targets longer ncRNAs and pre-mRNAs, which are polyadenylated by PAPOLA/PAPOLG, via the PPD and/or PAXT pathways. PAXT can also interact with the CBC via ARS2 and ZC3H18. The human TRAMP-like complexes, composed of the RNA-binding protein ZCCHC7, MTR4 and one of the pA-polymerases PAPD5 or PAPD7, mediate the decay and processing of nucleolar rRNAs.

Figure 3.

Schematic overview of S. cerevisiae TRAMP4/5 complexes and their physical relationships with the NNS and exosome complexes. TRAMP4 and TRAMP5 can both be recruited to their targets via Air1/2-mediated RNA binding. In addition, TRAMP4 recruitment to some ncRNAs is enhanced by the direct binding of Trf4 to the Nrd1 subunit of the NNS complex. TRAMP4/5 complexes contact the exosome via Mtr4. TRAMP4 interaction with the exosome can be further enhanced via binding of Nab3 to Rrp6 [83].