The hunt for the 3^′ endonuclease

Abstract

Pre-mRNAs are typically processed at the 3(') end by cleavage/polyadenylation. This is a two-step processing reaction initiated by endonucleolytic cleavage of pre-mRNAs downstream of the AAUAAA sequence or its variant, followed by extension of the newly generated 3(') end with a poly(A) tail. In metazoans, replication-dependent histone transcripts are cleaved by a different 3(') end processing mechanism that depends on the U7 small nuclear ribonucleoprotein and the polyadenylation step is omitted. Each of the two mechanisms occurs in a macromolecular assembly that primarily functions to juxtapose the scissile bond with the 3(') endonuclease. Remarkably, despite characterizing a number of processing factors, the identity of this most critical component remained elusive until recently. For cleavage coupled to polyadenylation, much needed help was offered by bioinformatics, which pointed to CPSF-73, a known processing factor required for both cleavage and polyadenylation, as the possible 3(') endonuclease. In silico structural analysis indicated that this protein is a member of the large metallo-β-lactamase family of hydrolytic enzymes and belongs to the β-CASP subfamily that includes several RNA and DNA-specific nucleases. Subsequent experimental studies supported the notion that CPSF-73 does function as the endonuclease in the formation of polyadenylated mRNAs, but some controversy still remains as a different cleavage and polyadenylation specificity factor (CPSF) subunit, CPSF-30, displays an endonuclease activity in vitro while recombinant CPSF-73 is inactive. Unexpectedly, CPSF-73 as the 3(') endonuclease in cleavage coupled to polyadenylation found a strong ally in U7-dependent processing of histone pre-mRNAs, which was shown to utilize the same protein as the cleaving enzyme. It thus seems likely that these two processing reactions evolved from a common mechanism, with CPSF-73 as the endonuclease.

A Complex Containing the CPSF73 Endonuclease and Other Polyadenylation Factors Associates with U7 snRNP and Is Recruited to Histone Pre-mRNA for 3′-End Processing

“…With the exception of CPSF73, which contacts the cleavage site in histone pre-mRNA and functions as the 3= endonuclease (31,53), the role of the remaining CPSF subunits in the U7-dependent processing is largely unknown. CPSF100 is a noncatalytic homologue, binding partner, and perhaps a regulator of CPSF73 (33,54).…”

3′-End processing of histone pre-mRNAs in Drosophila: U7 snRNP is associated with FLASH and polyadenylation factors

“…What could be the nature and function of this factor? It may, for example, represent a hypothetical activator of the CPSF73 endonuclease, which was speculated to depend on a structural rearrangement prior to acquiring the catalytic activity (Mandel et al 2006;Dominski 2010). Alternatively, it may function by bypassing the requirement for CstF64 or Ars2 in 3 ′ -end processing in Drosophila.…”

Delineating the Structural Blueprint of the Pre-mRNA 3′-End Processing Machinery

“…A CPSF subunit that has gained considerable attention is CPSF-73, largely because it turns out to be the endonuclease that has been sought for 3 decades (40). The earliest clue to its function came from a sequence analysis showing that CPSF-73 belongs to the metallo-␤-lactamase (M␤L) superfamily, whose members are mostly hydrolases dependent on metal ions (41).…”

“…Symplekin tightly associates with CPSF-73 and CPSF-100 (156,157), forming a shared stable core complex for both general and histone pre-mRNA 3= processing (157). As a consequence, it has been speculated that symplekin may regulate the nuclease activity of CPSF-73 through direct interactions or by recruiting additional regulatory factors (40,157), but further investigation is necessary to test this hypothesis.…”