Review
Finishing touches: Post-translational modification of protein factors involved in mammalian pre-mRNA 3′ end formation

https://doi.org/10.1016/j.biocel.2008.03.016Get rights and content

Abstract

In eukaryotes, a pre-messenger RNA (pre-mRNA) must undergo several processing reactions before it is exported to the cytoplasm for translation. One of these reactions, endonucleolytic 3′ cleavage at the polyadenylation site, prepares the pre-mRNA for addition of the poly(A) tail and defines the 3′ untranslated region (UTR), which typically contains important gene expression regulatory sequences. While the protein factors responsible for the endonucleolytic cleavage have been largely identified, the means by which their action is limited to the 3′ end of the transcription unit and coordinated with other co-transcriptional events remains unclear. In this review, we summarize and review recent findings revealing that the mammalian 3′ cleavage factors undergo extensive post-translational modification. These modifications include: arginine methylation, lysine sumoylation, lysine acetylation, and the phosphorylation of serine, threonine and tyrosine residues. Every cleavage factor, though not every subunit, is affected. Human Fip1 and the 59 kDa subunit of cleavage factor I emerge as the most frequently modified core cleavage factor subunits. We outline and compare the various proteomic methods that have uncovered these modifications, and review emerging hypotheses concerning their function. The roles of these covalent but reversible modifications in other systems suggest that 3′ end formation in mammals relies upon post-translational modification for proper function and regulation.

Introduction

In eukaryotes, nearly all protein-encoding pre-messenger RNAs (pre-mRNAs) undergo a series of processing reactions before they are exported to the cytoplasm for translation. These reactions, now understood to occur for the most part co-transcriptionally, include the addition of the 5′-cap structure, removal of non-coding introns by the splicesome, 3′ cleavage and polyadenylation, and mRNA editing. The 3′ cleavage at the polyadenylation site (pA site) cuts the nascent mRNA into two fragments, producing the 3′ end of the templated portion of the transcript in preparation for the addition by poly(A) polymerase (PAP) of the untemplated poly(A) tail (Fig. 1). The cleavage reaction therefore defines the 3′ untranslated region (3′ UTR), the regulatory element-rich segment of the transcript that lies between the translation stop codon and the poly(A) tail. Many human genes are subject to cleavage and polyadenylation at multiple pA sites (Edwalds-Gilbert et al., 1997, Tian et al., 2005), and so can possess variant 3′ UTRs and, in some cases, altered C-terminal protein sequences, though little is known about how regulatory choices are made among the different cleavage sites.

Many protein cleavage factors contribute to the recognition and endonucleolytic cleavage of the pA site in mammalian cells. For a recent review see Mandel et al. (2007). These factors include the cleavage and polyadenylation specificity factor (CPSF), which recognizes the highly conserved poly(A) signal (PAS) hexamer, A(A/U)UAAA, located 10–30 nucleotides (nt) upstream of the cleavage site, the cleavage stimulation factor (CstF), which recognizes the less precisely conserved U- or G/U-rich downstream element (DSE) generally found within 30 nt downstream of the cleavage site, cleavage factors I (CFIm) and II (CFIIm), symplekin, PAP, and the RNA Polymerase II C-terminal domain (CTD). These factors are shown as a hypothetical pre-cleavage complex in Fig. 2.

Though most of the protein factors responsible for the endonucleolytic cleavage reaction have been identified, exactly how their action is limited to the 3′ end of the transcription unit and coordinated with other co-transcriptional events, including transcription termination, is poorly understood. Recent in vitro evidence indicates that selective post-translational modification (PTM) of the 3′ cleavage factors may influence the control and coordination of 3′ cleavage during each transcription cycle. PTMs may be looked upon as the finishing touches put on certain proteins to aid in their function or regulation, while 3′ cleavage and polyadenylation constitutes another sort of finishing touch on the newly minted pre-mRNA. In this review, we focus on recent findings demonstrating that the mammalian 3′ cleavage factors are modified by a variety of covalent PTMs, many of which have been brought to light by large unbiased proteomic mass spectrometry (MS) screens. Alcohol residue phosphorylation, lysine acetylation, arginine methylation, and lysine sumoylation have all been found among the various 3′ cleavage factors. Every cleavage factor, though not every subunit, is affected. We also compile, summarize and discuss these modifications and the different contexts in which they have been identified. We begin with phosphorylation, the most widespread modification. We review the various phosphoproteomic approaches that have uncovered phosphate-modified cleavage factor residues, and then compile phosphorylation sites by cleavage factor. Also described are recent discoveries of cleavage factor acetylation, methylation and sumoylation. Where possible, we consider emerging hypotheses regarding the function of these modifications in mammalian pre-mRNA 3′ end formation.

Section snippets

Phosphorylation

Reversible phosphorylation is the most common post-translational protein modification in eukaryotes. It is thought to control some aspect of almost all cellular processes (Hubbard and Cohen, 1993), a scope consistent with the large number of mammalian genes that encode kinases and phosphatases (Cohen, 2002). Phosphorylation can be used by a cell to control a protein in many ways, from switching on or off a particular enzymatic activity, to modulating protein–protein interactions, intracellular

Lysine acetylation

Whereas Ser/Thr/Tyr phosphorylation creates from a neutral side chain one having a −2 charge, lysine acetylation neutralizes the +1 charge found on the primary amino group of this amino acid at physiological pH. This modification has been most often studied in the context of the chromatin histone proteins, but other proteins undergo acetylation as well, and more continue to be found (Kouzarides, 2000). Proteomic screens for acetylation are less numerous than for phosphorylation. One proteomic

Arginine methylation

In contrast to phosphorylation and acetylation, arginine methylation does not alter the charge of the affected amino acid. It does however increase the size of the arginine side chain, reduce the number of its hydrogen bond donors by the number of methyl groups added, and make the residue more hydrophobic. Methyl groups are added to arginine residues by the protein arginine methyltransferases (PRMTs) in either a symmetric or asymmetric arrangement. For recent reviews of this large family of

Lysine sumoylation

The only other PTM that has been documented within the cleavage factors is the SUMO (small ubiquitin-related modifier) group (Vethantham et al., 2007), recently reviewed in reference (Geiss-Friedlander and Melchior, 2007). The human genome encodes four distinct SUMOs, designated SUMO-1 to SUMO-4 (Melchior, 2000). Though small by protein standards at about 10 kDa, the SUMO protein is relatively large compared to the other cleavage factor PTMs, and consequently alters a protein's activity mainly

Reversible post-translational modification: how might it regulate 3′ cleavage?

In vitro RNA processing assays carried out in the absence of transcription will continue to be useful for understanding the function of PTMs, but most pre-mRNA 3′ processing is thought to take place while the nascent RNA is still attached to the elongating, though likely paused, RNA Pol II (Bentley, 2005, Glover-Cutter et al., 2008, Hirose and Manley, 2000, Rigo et al., 2005). Several of the 3′ cleavage factors are recruited to the elongating RNA Pol II complex long before they are needed for

Summary and outlook

We have reviewed here the methods used to discover PTMs within the human 3′ pre-mRNA cleavage factors, and catalogued the known sites of phosphorylation, acetylation, methylation and sumoylation. Post-translational modification adds to the energy cost of making a functional protein, so it is reasonable to propose that there is a functional purpose for creating the modifications in most cases, and we hypothesize that the reasons here may involve 3′ cleavage activity regulation. Preliminary in

Acknowledgements

This work was supported by the City College of New York Science Division. We also acknowledge NIH grant R21GM073944 to K.R. and the NIH Research Centers in Minority Institutions grant 5G12RR03060. D.L.V.B. is a Barry M. Goldwater Scholarship and Intel Science Talent Search award recipient and gratefully acknowledges their support. We thank G. Martin and W. Keller for sharing unpublished results, and B. Blagoev and B. Tian for helpful discussions.

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