A CPSF-73 Homologue Is Required for Cell Cycle Progression but Not Cell Growth and Interacts with a Protein Having Features of CPSF-100

Abstract

Formation of the mature 3 ends of the vast majority of cellular mRNAs occurs through cleavage and polyadenylation and requires a cleavage and polyadenylation specificity factor (CPSF) containing, among other proteins, CPSF-73 and CPSF-100. These two proteins belong to a superfamily of zinc-dependent ␤-lactamase fold proteins with catalytic specificity for a wide range of substrates including nucleic acids. CPSF-73 contains a zinc-binding histidine motif involved in catalysis in other members of the ␤-lactamase superfamily, whereas CPSF-100 has substitutions within the histidine motif and thus is unlikely to be catalytically active. Here we describe two previously unknown human proteins, designated RC-68 and RC-74, which are related to CPSF-73 and CPSF-100 and which form a complex in HeLa and mouse cells. RC-68 contains the intact histidine motif, and hence it might be a functional counterpart of CPSF-73, whereas RC-74 lacks this motif, thus resembling CPSF-100. In HeLa cells RC-68 is present in both the cytoplasm and the nucleus whereas RC-74 is exclusively nuclear. RC-74 does not interact with CPSF-73, and neither RC-68 nor RC-74 is found in a complex with CPSF-160, indicating that these two proteins form a separate entity independent of the CPSF complex and are likely involved in a pre-mRNA processing event other than cleavage and polyadenylation of the vast majority of cellular pre-mRNAs. RNA interference-mediated depletion of RC-68 arrests HeLa cells early in G 1 phase, but surprisingly the arrested cells continue growing and reach the size typical of G 2 cells. RC-68 is highly conserved from plants to humans and may function in conjunction with RC-74 in the 3 end processing of a distinct subset of cellular pre-mRNAs encoding proteins required for G 1 progression and entry into S phase.In metazoans, there are two distinct mechanisms of 3Ј end processing of pre-mRNAs that lead to formation of mature mRNAs. The vast majority of pre-mRNAs are processed at the 3Ј end by a coupled cleavage and polyadenylation reaction (9,57,71,78). However, the replication-dependent histone premRNAs are processed by a one-step mechanism that involves only a cleavage reaction (13,40,47). Both mechanisms require two cis-acting elements in the pre-mRNA and involve different sets of trans-acting factors.In mammalian pre-mRNAs, the cleavage and polyadenylation reaction occurs about 20 nucleotides downstream of a highly conserved polyadenylation signal, AAUAAA. The AA UAAA sequence is recognized by cleavage and polyadenylation specificity factor 160 (CPSF-160) (32, 49), which exists in a stable complex with three other proteins, CPSF-100, CPSF-73, and CPSF-30 (2, 4, 31, 33, 48). The CPSF complex also contains a fifth component, Fip1, which may be only loosely associated with the other subunits or which may exist in nonequimolar amounts in the complex (34). The CPSF complex is required for both cleavage and polyadenylation. About 30 nucleotides downstream of the cleavage site there is a weakly conserved GU-rich element, which is recogni...

Protein factors in pre-mRNA 3′-end processing

“…Sequence analysis suggests that a leucine zipper, which is usually involved in protein-protein interactions, may be present in the final 30 residues of Brr5p/Ysh1p. The C-terminus of CPSF-73 does interact with other proteins in the 3′-end processing complex, including CPSF-100 [117] and Pta1p [111]. Brr5p/Ysh1p has also been shown to interact strongly with Clp1p, but the binding region is unknown [118].…”

“…CPSF-73 has a second isoform in humans, known as RC-68 or Int9 [117,120]. It may be involved in 3′-end processing of small nuclear RNAs and it interacts with the second isoform of CPSF-100, RC-74 or Int11 [117,120].…”

“…It may be involved in 3′-end processing of small nuclear RNAs and it interacts with the second isoform of CPSF-100, RC-74 or Int11 [117,120].…”

“…In contrast, the full-length protein is required for interactions with the other proteins [118]. Yeast two-hybrid studies showed that the last 245 residues of CPSF-73 can interact with CPSF-100 [117]. Besides protein-protein interactions, Cft2p/Ydh1p can bind the pre-mRNA near the cleavage site [72,122].…”

“…Like CPSF-73, CPSF-100 has a second isoform in humans, known as RC-74 or Int11, also with disrupted zinc binding sites [117,120].…”