The 3′ End Formation of Histone mRNA Requires U7 snRNA

KEY CONCEPTS
- The expression of histone mRNAs is replication dependent and is regulated during the cell cycle.
- Histone mRNAs are not polyadenylated; their 3′ ends are generated by a cleavage reaction that depends on the structure of the mRNA.
- The cleavage reaction requires the stem-loop binding protein (SLBP) to bind to a stem-loop structure and the U7 snRNA to pair with an adjacent single-stranded region.
- The cleavage reaction is catalyzed by a factor shared with the polyadenylation complex.
Biogenesis of the canonical histones is primarily controlled by the regulation of histone mRNA abundance during the cell cycle. At this G1/S transition, the abundance of histone mRNAs is increased more than 30-fold due to elevated transcription; this process is regulated by the cyclin E/Cdk2 complex . The rise in histone mRNAs is followed by a rapid decay of histone mRNAs at the end of S phase.
Canonical histone mRNAs are not polyadenylated (except in S. cerevisiae). (Note that some of the histone variants, such as H3.3, are not cell-cycle regulated and are polyadenylated.) The formation of their 3′ ends is therefore different from that of the coordinated cleavage/polyadenylation reaction; it depends upon a highly conserved stem-loop structure located 14 to 50 bases downstream from the termination codon and a histone downstream element (HDE) located about 15 nucleotides downstream of the stem-loop. Cleavage occurs between the stem-loop and HDE, leaving five bases downstream of the stem-loop. Mutations that prevent formation of the duplex stem of the stem-loop prevent formation of the end of the RNA.
Secondary mutations that restore duplex structure (though not necessarily the original sequence) restore 3′ end formation. This indicates that formation of the secondary structure is more important than the exact sequence.
The reaction forming the histone 3′ end is shown in FIGURE 1. Two factors are required to specify the cleavage reaction: The stem-loop binding protein (SLBP) recognizes the stem-loop structure, and the 5′ end of U7 snRNA base pairs with a purine-rich sequence within HDE. U7 snRNP is a minor snRNP consisting of the 63-nucleotide U7 snRNA and a set of several proteins related to snRNPs involved in mRNA splicing . Unique to U7 snRNP are two Sm-like proteins, LSM10 and LSM11, which replace Sm D1 and D2 in the splicing snRNPs. Prevention of base pairing between U7 snRNA and HDE impairs 3′ processing of the histone mRNAs, and compensatory mutations in U7 snRNA that restore complementarity restore 3′ processing. This indicates that U7 snRNA functions by base pairing with the histone mRNAs.

FIGURE 2.Generation of the 3′ end of histone h3 mRNA depends on a conserved hairpin and a sequence that base pairs with U7 snRNA.
Cleavage to generate a 3′ terminus occurs at a fixed distance from the site recognized by U7 snRNA, which suggests that the snRNA is involved in defining the cleavage site. The factor responsible for cleavage is a specific cleavage and polyadenylation specificity factor (CPSF73). Thus, this member of the metallo-β-lactamase family plays a key role in 3′ end formation for both polyadenylated mRNAs and nonpolyadenylated histone mRNAs. Several other proteins have been identified as important for histone 3′ end formation, including CPSF100 and Symplekin, but their specific roles remain to be defined. These additional proteins may provide scaffold functions to stabilize the 3′-end–processing complex.
Interestingly, disruption of U7 base pairing with the target sequences in histone genes or siRNA-mediated depletion of other components involved in the formation of the histone 3′ end all result in transcriptional readthrough and polyadenylation by using a poly(A) signal downstream from the DHE. Thus, similar to the role of mRNA cleavage/polyadenylation in Pol II transcriptional termination on most protein-coding genes, U7-mediated RNA cleavage during 3′ end formation appears to be critical for transcriptional termination on histone genes.