Research Spotlight: Mechanism for 25 kDa subunit of human cleavage factor Im recognition of UGUA RNA sequence element

By Matt Wargo • October 6th, 2010

Citation: Yang Q, Gilmartin GM, and Doublié S. 2010. Structural basis of UGUA recognition by the Nudix protein CFIm25 and implications for a regulatory role in mRNA 3’ processing. Proc Natl Acad Sci, 107, 10062-7.

Direct link to the publication at the PNAS website

Authors’ Association with MMG:
Qin Yang – is a graduate student in the MMG Department
Sylvie Doublié – is a Professor in the MMG Department
Gregory M. Gilmartin – is an Associate Professor in the MMG Department

Where we started
In eukaryotes, 3’ cleavage and polyadenylation, together with splicing and 5’ capping, are processing steps that most messenger RNA precursors (pre-mRNA) undergo before being transported from the nucleus to the cytoplasm for translation. Cleavage factor Im (CFIm), a two subunit complex, is essential for the efficiency of the 3’ cleavage reaction and the selection of the site where cleavage takes place. The function of CFIm is mediated by the recognition of specific sequence elements on the pre-mRNA. The UGUA sequence preference and its importance in 3’ processing have been shown by SELEX [1] and biochemical experiments [1, 2], but the subunit responsible for UGUA recognition had not been identified yet.

What we did
This paper demonstrated that the smaller 25 kDa subunit (CFIm25) is the subunit that recognizes the UGUA sequence specifically. A detailed mechanism for recognition was illustrated by two crystal structures of CFIm25 in complex with 6-mer RNA oligonucleotides containing a UGUA element. The binding properties of protein and RNA sequence variants were analyzed and confirmed the structural data.

What we learned
The structure uncovered a new sequence specific RNA binding domain. The only predicted domain of CFIm25 is a Nudix domain, which usually hydrolyzes (di)nucleotides. Previous work done in the lab had shown that although CFIm25 harbors a Nudix fold and is able to bind diadenosine tetraphosphate (Ap4A), it has no detectable hydrolase activity [3]. The crystal structures of the RNA-bound protein revealed that the Nudix fold has been transformed into a platform for the sequence-specific binding of single-stranded RNA.
CFIm25 forms a homodimer in solution [3]. Our structural model and biochemistry data indicate that a CFIm25 dimer can bind two UGUA elements simultaneously, which is consistent with the prevalence of multiple UGUA elements in the 3’ UTR region. This finding provides a basis for how CFIm may modulate alternative cleavage site selection. The structure also revealed that the binding of RNA and the signaling molecule Ap4A are mutually exclusive, suggesting that small molecules may play a role in the regulation of mRNA processing.

1. Brown KM & Gilmartin GM (2003) A mechanism for the regulation of pre-mRNA 3′ processing by human cleavage factor Im. Mol Cell 12(6):1467-1476.

2. Venkataraman K, Brown KM, & Gilmartin GM (2005) Analysis of a noncanonical poly(A) site reveals a tripartite mechanism for vertebrate poly(A) site recognition. Genes Dev 19(11):1315-1327.

3. Coseno M, et al. (2008) Crystal structure of the 25 kDa subunit of human cleavage factor Im. Nucleic Acids Res 36(10):3474-3483.

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