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The hunt for the 3′ endonuclease

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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. Copyright © 2010 John Wiley & Sons, Ltd. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes RNA Processing > 3' End Processing

3 end processing by cleavage/polyadenylation. A tentative arrangement of major factors involved in cleavage coupled to polyadenylation. Some of the less characterized components and subunits are omitted for simplicity. The thick black line represents the pre‐mRNA substrate containing the AAUAAA and GU‐rich sequences. The RNA‐binding subunits of the cleavage and polyadenylation specificity factor (CPSF) and cleavage stimulation factor complexes, CPSF‐160 and CstF‐64, interact with the AAUAAA and GU‐rich elements, respectively. The substrate is cleaved between the two elements, downstream of the CA dinucleotide. The reaction is catalyzed by the 73 kDa subunit of CPSF, CPSF‐73, as indicated by a dashed arrow. All three subunits of CstF are shown as a dimer.

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The endo‐ and 5 exonuclease activities of CPSF‐73 analyzed in mouse nuclear extracts using a set of truncated histone pre‐mRNAs. (a) CPSF‐73 endonucleolytically cleaves histone pre‐mRNA five nucleotides downstream of the stem‐loop and the downstream cleavage product containing the histone downstream element (HDE; underlined) is subsequently degraded by the 5 exonuclease activity of the same protein. The latter activity releases the U7 snRNP for another round of processing. (b) 5 Truncated pre‐mRNA substrates containing the HDE (underlined) are either cleaved endonucleolytically (large vertical arrows) or degraded in the 5 → 3 direction (large horizontal arrowheads) by the two different activities of CPSF‐73, depending on the number of nucleotides upstream of the HDE. Small vertical arrows indicate minor endonucleolytic cleavage sites in the HDE + 16/m RNA in which the 5 end is modified with a 2‐O‐methyl group (m). Substrates containing seven or fewer nucleotides upstream of the HDE efficiently bind the U7 snRNP but are resistant to either reaction, possibly due to the inability to contact Lsm10.

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A universal module that functions as the 3 endonuclease in two distinct processing complexes. CPSF‐73, CPSF‐100, and symplekin are shared by both cleavage/polyadenylation and 3 end processing of histone pre‐mRNAs. Symplekin and CPSF‐100 may function together as a binding platform that interacts with various components of the two respective processing complexes, whereas CPSF‐73 is the catalytic component that functions as the endonuclease. At the final stage of processing, this basic module may also include a putative CPSF‐73 activator that converts the latent form of CPSF‐73 into an active nuclease. Alternatively, the activation may be mediated by the correct positioning of all protein factors and the RNA substrate in the processing complex that collectively promotes a conformational change in CPSF‐73.

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Residues of human CPSF‐73 involved in zinc binding and catalysis. (a) Domain organization of the N‐terminal region of human CPSF‐73 (amino acids 1–460). The β‐CASP domain begins with amino acid 209 and ends with amino acid 394. Conserved amino acids in the single‐letter code are shown on the top of the chart and their positions and motif assignments are shown below. Down and up arrows indicate residues involved in the coordination of Zn1 and Zn2, respectively. The double head arrow indicates the residue bridging the two zinc atoms. (b) The octahedral coordination sphere that binds the two zinc ions in human CPSF‐73.

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3 end processing of replication‐dependent histone pre‐mRNAs. A tentative arrangement of known factors assembled on metazoan histone pre‐mRNA (thick black line). The unique proteins of the U7 small nuclear ribonucleoprotein (snRNP), Lsm10, and Lsm11 are indicated in gray. Stem‐loop binding protein interacts with the stem‐loop and makes additional contacts with ZFP100, which in turn interacts with the U7 snRNP. The 5 terminal region of the U7 snRNA (thick gray line) base pairs (vertical lines) with the histone downstream element in histone pre‐mRNA. This interaction brings SmD3, SmB, and Lsm10 of the U7 Sm complex to the substrate RNA, with the three proteins acting as the molecular ruler to determine the site of cleavage (indicated by an arrow). FLASH interacts with the N‐terminal region of Lsm11 and may directly or indirectly recruit symplekin, CPSF‐100, and CPSF‐73 (the endonuclease). The latter three proteins are shared with cleavage/polyadenylation.

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RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes
RNA Processing > 3′ End Processing

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