The hunt for the 3' endonuclease

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.