Structural basis of UGUA recognition by the Nudix protein CFI _m 25 and implications for a regulatory role in mRNA 3′ processing

Abstract

Human Cleavage Factor Im (CFI m ) is an essential component of the pre-mRNA 3′ processing complex that functions in the regulation of poly(A) site selection through the recognition of UGUA sequences upstream of the poly(A) site. Although the highly conserved 25 kDa subunit (CFI m 25) of the CFI m complex possesses a characteristic α/β/α Nudix fold, CFI m 25 has no detectable hydrolase activity. Here we report the crystal structures of the human CFI m 25 homodimer in complex with UGUAAA and UUGUAU RNA sequences. CFI m 25 is the first Nudix protein to be reported to bind RNA in a sequencespecific manner. The UGUA sequence contributes to binding specificity through an intramolecular G:A Watson-Crick/sugar-edge base interaction, an unusual pairing previously found to be involved in the binding specificity of the SAM-III riboswitch. The structures, together with mutational data, suggest a novel mechanism for the simultaneous sequence-specific recognition of two UGUA elements within the pre-mRNA. Furthermore, the mutually exclusive binding of RNA and the signaling molecule Ap 4 A (diadenosine tetraphosphate) by CFI m 25 suggests a potential role for small molecules in the regulation of mRNA 3′ processing.T he transcriptome complexity of higher eukaryotes requires the coordinate recognition of an array of alternative pre-mRNA processing signals in a developmental and tissue-specific manner (1, 2). The sequences that direct pre-mRNA splicing and 3′ processing are initially recognized within the nascent transcript in a process that is intimately coupled to transcription (3, 4). While the recognition of exons within the pre-mRNA is mediated by both RNA:RNA and protein:RNA interactions (5), the 3′ processing of polyadenylated mRNAs appears to rely solely on the interaction of protein factors (6) with unstructured RNA sequences (7) within the nascent transcript.Vertebrate pre-mRNA 3′ processing signals are recognized by a tripartite mechanism through which a set of short RNA sequences direct the cooperative binding of three multimeric 3′ processing factors, cleavage factor I m (CFI m ), cleavage and polyadenylation specificity factor (CPSF), and cleavage stimulation factor (CstF) (8). CPSF and CstF bind the AAUAAA hexamer and downstream GU-rich elements that flank the poly(A) site, respectively, whereas CFI m interacts with upstream sequences that may function in the regulation of alternative polyadenylation (9-11). SELEX and biochemical analyses have identified the sequence UGUAN (N ¼ A > U > G, C) as the preferred binding site of CFI m (11). In this report we have taken a structural approach to determine the mechanism of sequence-specific RNA binding by CFI m .CFI m is composed of a large subunit of 59, 68, or 72 kDa and a small subunit of 25 kDa (CFI m 25, also referred to as CPSF5 or NUDT21) (12, 13), both of which contribute to RNA binding (14). The large subunit, encoded by either of two paralogs (CPSF6 and CPSF7), contains an N-terminal RNA Recognition Motif (RRM), an internal polyproline-rich region, and a C-termina...

Structural basis of pre-mRNA recognition by the human cleavage factor Im complex

“…As the molecular weights of CF I m 25 and CF I m 68RRM are 24 and 10 kDa, respectively, this result suggests that the CF I m 25-CF I m 68RRM complex exists as a heterotetramer in solution. The heterotetrameric state is also consistent with previous reports that two subunits of CF I m form a heterotetramer in solution [17]. Outlier (%) 0.1 …”

“…Recently, CF I m has been shown to be a heterotetramer in solution [17]. Our structure confirms that CF I m 25 [17] observed that the CF I m 25 dimer bound RNA containing two separated UGUAA elements with 100-fold higher affinity than RNA containing only one UGUAA element.…”

“…Our structure confirms that CF I m 25 [17] observed that the CF I m 25 dimer bound RNA containing two separated UGUAA elements with 100-fold higher affinity than RNA containing only one UGUAA element. The minimum distance between the two UGUA elements, which is required to observe this gain in affinity is five nucleotides.…”

“…The minimum distance between the two UGUA elements, which is required to observe this gain in affinity is five nucleotides. Unlike the structures of the UGUAAA-and UUGUAU-CF I m 25 complexes, which show CF I m 25 dimer binding with only one RNA molecule in the crystals [17], our structure shows that two UGUAA RNA sequences, with antiparallel orientation, bind to one CF I m 25-CF I m 68RRM heterotetramer, providing structural evidence to support the simultaneous binding of two UGUAA elements to the CF I m 25 dimer. The anti-parallel positioning of the two UGUAA elements and the discontiguous RNA-binding surfaces confirm the importance of the separation by a certain number of bases of the two UGUAA elements.…”

“…Yang et al [17] reported the structure of the CF I m 25-UGUAAA complex, which showed that the RNA hexamer was bound by one molecule of the CF I m 25 dimer and partially by an adjacent molecule in the crystal ( Figure 5C). In addition, the authors found that the CF I m 25 dimer can bind two UGUA elements in solution.…”