Efficient mRNA Polyadenylation Requires a Ubiquitin-Like Domain, a Zinc Knuckle, and a RING Finger Domain, All Contained in the Mpe1 Protein

Abstract

Almost all eukaryotic mRNAs must be polyadenylated at their 3= ends to function in protein synthesis. This modification occurs via a large nuclear complex that recognizes signal sequences surrounding a poly(A) site on mRNA precursor, cleaves at that site, and adds a poly(A) tail. While the composition of this complex is known, the functions of some subunits remain unclear. One of these is a multidomain protein called Mpe1 in the yeast Saccharomyces cerevisiae and RBBP6 in metazoans. The three conserved domains of Mpe1 are a ubiquitin-like (UBL) domain, a zinc knuckle, and a RING finger domain characteristic of some ubiquitin ligases. We show that mRNA 3=-end processing requires all three domains of Mpe1 and that more than one region of Mpe1 is involved in contact with the cleavage/polyadenylation factor in which Mpe1 resides. Surprisingly, both the zinc knuckle and the RING finger are needed for RNA-binding activity. Consistent with a role for Mpe1 in ubiquitination, mutation of Mpe1 decreases the association of ubiquitin with Pap1, the poly(A) polymerase, and suppressors of mpe1 mutants are linked to ubiquitin ligases. Furthermore, an inhibitor of ubiquitin-mediated interactions blocks cleavage, demonstrating for the first time a direct role for ubiquitination in mRNA 3=-end processing. Polyadenylation is an essential step in the production of functional eukaryotic mRNA that will be efficiently utilized in translation. It is a nuclear processing event that involves the cleavage of mRNA precursor followed by the addition of a poly(A) tail and is carefully coordinated with other events involved in mRNA synthesis and utilization, such as transcription, splicing, assembly of mRNA into ribonucleoprotein complexes, and mRNA export. Many recent studies have also revealed how selection of the cleavage site can globally affect the type of mRNA produced by cells (1-3). Polyadenylation requires a suite of multiple factors whose subunits are conserved across eukaryotic species (4-6). Significant progress has been made in understanding the contributions of each factor to the recognition of the poly(A) site, the execution of the processing steps, and regulation. However, the precise role of each subunit in this complex process and the impact of posttranslational modifications on the regulation of polyadenylation have not been completely defined.In Saccharomyces cerevisiae, the factors needed for mRNA 3=-end processing are the RNA-binding protein Hrp1 and two multisubunit complexes called cleavage/polyadenylation factor (CPF) and cleavage factor IA (CF IA). One poorly characterized but essential subunit of CPF identified more than 10 years ago is Mpe1, which is needed for both processing steps (7). The mammalian Mpe1 homolog, RBBP6, interacts with the tumor suppressor proteins p53 and pRb (8, 9), possibly linking polyadenylation to regulation of cell growth. Even though RBBP6 is found in the mammalian mRNA 3=-end processing complex (10), it has not been shown to function in polyadenylation.Mpe1 is a particularly interesting s...

RBBP6 isoforms regulate the human polyadenylation machinery and modulate expression of mRNAs with AU-rich 3′ UTRs

“…for RNA binding (Lee and Moore 2014). The same study also showed that Mpe1 is required for the previously established ubiquitination of PAP (Mizrahi and Moore 2000) and for polyadenylation activity.…”

E11/Podoplanin Protein Stabilization Through Inhibition of the Proteasome Promotes Osteocyte Differentiation in Murine in Vitro Models

“…In other studies MG132 and Bortezomib have been reported to induce mRNA increases through both transcriptional (promoter activation) and post‐transcriptional (mRNA stability) mechanisms (Butler et al, ; Laroia et al, ; Shimizu et al, ). Conversely, MG132 causes defective polyadenylation (Lee and Moore, ) and whilst it has been previously shown that in human tissues, there is a 2.7 kb and a 0.9 kb E11 mRNA species, which differ in their polyadenylation (Martin‐Villar et al, ), there is no suggestion that this is the case in murine tissues. It would be interesting however to examine whether the stabilization of E11 observed here is maintained upon addition of a protein synthesis inhibitor, such as cycloheximide.…”

The end of the message: multiple protein–RNA interactions define the mRNA polyadenylation site

“…The yeast mRNA 3 ′ processing factor CPF contains Mpe1 (Rbbp6 homolog) (see below), Pap1, Pta1 (Symplekin), and the homologs of all six subunits of the mammalian CPSF complex, including Cft1 (CPSF160), Cft2 (CPSF100), Ysh1/Brr5 (CPSF73), Fip1 (Fip1), Yth1 (CPSF30), and Pfs2 (Wdr33) (Zhao et al 1999). Similar to CPSF, multiple CPF subunits have been shown to bind RNA, including Cft1, Cft2, Fip1, Yth1, and Mpe1 (Barabino et al 1997;Zhao et al 1997;Dichtl et al 2002;Lee and Moore 2014).…”

“…This N-terminal region, which is the region of similarity with its yeast homolog, Mpe1, is sufficient for 3 ′ processing activity in vitro. In both proteins, the zinc knuckle and RING domains function in RNA binding, although in neither case does binding appear sequence-specific (Di Giammartino et al 2014;Lee and Moore 2014). Interestingly, knockdown of Rbbp6 in mammalian cells preferentially inhibits the 3 ′ processing of mRNAs that contain AU-rich elements (AREs) in their 3 ′ UTRs (Di Giammartino et al 2014).…”

“…For example, Mpe1 has been implicated in regulating ubiquitination of PAP and potentially other factors (Lee and Moore 2014). Rbbp6 has also been shown to possess E3 ligase activity and function as an activator of Mdm2-mediated ubiquitination of p53 (Li et al 2007;Miotto et al 2014).…”