3'-End Processing of Eukaryotic mRNA: Machinery, Regulation, and Impact on Gene Expression

Abstract

Formation of the 3' end of a eukaryotic mRNA is a key step in the production of a mature transcript. This process is mediated by a number of protein factors that cleave the pre-mRNA, add a poly(A) tail, and regulate transcription by protein dephosphorylation. Cleavage and polyadenylation specificity factor (CPSF) in humans, or cleavage and polyadenylation factor (CPF) in yeast, coordinates these enzymatic activities with each other, with RNA recognition, and with transcription. The site of pre-mRNA cleavage can strongly influence the translation, stability, and localization of the mRNA. Hence, cleavage site selection is highly regulated. The length of the poly(A) tail is also controlled to ensure that every transcript has a similar tail when it is exported from the nucleus. In this review, we summarize new mechanistic insights into mRNA 3'-end processing obtained through structural studies and biochemical reconstitution and outline outstanding questions in the field.

Keywords: endonuclease; poly(A) tail; polyadenylation; polymerase; pre-mRNA processing; transcription.

Figure 1

3′-end processing depends on multiple protein complexes and cis-regulatory elements. Schematic representations of cotranscriptional (a) cleavage and (b) polyadenylation reactions catalyzed by CPSF and regulated by accessory factors. (c) The pre-mRNA region carrying the cis-regulatory elements required for 3′-end processing. Protein factors binding to each element in yeast (top) and humans (bottom) are indicated. (d) Schematics of the proteins required for 3′-end processing. Panel c adapted from Reference 7; panel d adapted from Reference 10. Abbreviations: CF, cleavage factor; CPF, cleavage and polyadenylation factor; CPSF, cleavage and polyadenylation specificity factor; CStF, cleavage stimulatory factor; CTD, C-terminal domain; mCF, mammalian cleavage factor; mPSF, mammalian polyadenylation specificity factor; PAS, polyadenylation signal; Pol II, RNA polymerase II; poly(A), polyadenosine; pre-mRNA, precursor messenger RNA.

Figure 2

mPSF/polymerase module specifically recognizes the pre-mRNA. (a,b) Overall architecture of (a) human mPSF bound to an RNA containing a PAS sequence (PDB ID: 6DNH) and (b) the budding yeast polymerase module bound to PAS RNA and Mpe1 (PDB ID: 7ZGR) (21, 33). (c,d) Close-up view of the PAS RNA-binding sites of (c) human mPSF and (d) the yeast polymerase module. Some hydrogen bonds between the RNA and the protein subunits are indicated by dashed black lines. Hydrogen bonds that mediate Hoogsteen base pairing between U3 and A4 in the human complex are depicted in dashed blue lines. Some protein residues that make hydrophobic and stacking interactions with the RNA are also shown in stick representation. Zinc ions bound to ZnF domains of CPSF30/Yth1 are shown in gray. Abbreviations: CPSF, cleavage and polyadenylation specificity factor; mPSF, mammalian polyadenylation specificity factor; PAS, polyadenylation signal; PDB ID, Protein Data Bank identifier; ZnF, zinc finger.

Figure 3. Activation of 3′processing endonucleases requires accessory factors.

(a) Structural comparison between the inactive state of CPSF73 (PDB ID: 2I7T) (38) and activated CPSF73 (PDB ID: 6V4X) (45) bound to Lsm10 and Lsm11 within the HCC. Lsm10 acts as a wedge that induces a rotation of the MβL relative to the β-CASP domain, opening the active site of the endonuclease. (b) Structural model of activated CPSF73 bound to RBBP6, which is based on the experimental structure of the yeast dimeric complex (PDB ID: 6I1D) (12) and the HCC (PDB ID: 6V4X) (45). (c) Structural comparison between the inactive state of INTS11 (PDB ID: 7BFP) (43) within the Integrator cleavage module and activated INTS11 within Integrator bound to the paused Pol II complex (PDB ID: 7PKS) (62). Similarly to Lsm10, SPT5 promotes opening of the active site. Abbreviations: CPSF, cleavage and polyadenylation specificity factor; HCC, histone cleavage complex; MβL, metallo-β-lactamase domain; PDB ID, Protein Data Bank identifier; Pol II, RNA polymerase II.

Figure 4. Eukaryotic pre-mRNA 3‘-end processing is tightly regulated and coordinated with splicing and transcription termination.

(a) Schematic representation of APA showing the factors that influence the choice of cleavage site. (b) Schematic representation of transcription termination. Red arrows depict phosphatase activity of CPSF/CPF acting on the CTD of Pol II and on transcription elongation factor SPT5. (c) Schematic representation of coupling between splicing, 3′-end processing, and transcription termination. Panel b adapted from Reference 93. Abbreviations: APA, alternative polyadenylation; CPF, cleavage and polyadenylation factor; CPSF, cleavage and polyadenylation specificity factor; CTD, C-terminal domain; PAS, polyadenylation signal; Pol II, RNA polymerase II; RBP, RNA-binding protein; TSS, transcription start site.

Figure 5

Models of the architecture of the eukaryotic 3‘-end processing machinery before activation, in its active cleavage state, and during polyadenylation. The order in which individual protein factors assemble is unknown. Abbreviations: CF, cleavage factor; CPSF, cleavage and polyadenylation specificity factor; CStF, cleavage stimulatory factor; mCF, mammalian cleavage factor; mPSF, mammalian polyadenylation specificity factor; PAP, poly(A) polymerase; PAS, polyadenylation signal.