An assembly process involving sequential recruitment of components and hierarchical dependency drives formation of the nuclear structures known as histone locus bodies.

SUMMARY 3′ end processing of histone pre-mRNA requires U7 snRNP, which binds downstream of the cleavage site and recruits the endonuclease CPSF-73. U7 snRNP contains a unique Sm ring in which the canonical SmD2 protein is replaced by Lsm11. We used the yeast two-hybrid system to identify binding partners of Lsm11 and selected the pro-apoptotic protein FLASH. Human FLASH interacts with Lsm11 in vitro and stimulates 3′ end processing of histone pre-mRNA in mammalian nuclear extracts. We also identified the FLASH ortholog in Drosophila and demonstrate that it interacts with Lsm11 in vitro and in vivo. Drosophila FLASH localizes to histone locus bodies and its depletion in fly cells inhibits U7-dependent processing resulting in polyadenylation of histone mRNAs. These results demonstrate that FLASH is an essential factor required for 3′ end maturation of histone mRNAs in both vertebrates and invertebrates and suggest a potential link between this process and apoptosis.

Proper gene expression relies on a class of ubiquitously expressed, uridine-rich small nuclear RNAs (snRNAs) transcribed by RNA polymerase II (RNAPII). Vertebrate snRNAs are transcribed from a unique promoter, which is required for proper 3-end formation, and cleavage of the nascent transcript involves the activity of a poorly understood set of proteins called the Integrator complex. To examine 3-end formation in Drosophila melanogaster, we developed a cell-based reporter that monitors aberrant 3-end formation of snRNA through the gain in expression of green fluorescent protein (GFP). We used this reporter in Drosophila S2 cells to determine requirements for U7 snRNA 3-end formation and found that processing was strongly dependent upon nucleotides located within the 3 stem-loop as well as sequences likely to comprise the Drosophila equivalent of the vertebrate 3 box. Substitution of the actin promoter for the snRNA promoter abolished proper 3-end formation, demonstrating the conserved requirement for an snRNA promoter in Drosophila. We tested the requirement for all Drosophila Integrator subunits and found that Integrators 1, 4, 9, and 11 were essential for 3-end formation and that Integrators 3 and 10 may be dispensable for processing. Depletion of cleavage and polyadenylation factors or of histone pre-mRNA processing factors did not affect U7 snRNA processing efficiency, demonstrating that the Integrator complex does not share components with the mRNA 3-end processing machinery. Finally, flies harboring mutations in either Integrator 4 or 7 fail to complete development and accumulate significant levels of misprocessed snRNA in the larval stages.In eukaryotes, the major transcripts produced by RNA polymerase II (RNAPII) include the polyadenylated [poly (A) ϩ ] mRNAs, the replication-dependent histone mRNAs, and the Sm class of small nuclear RNAs (snRNAs). The 3Ј ends of these three general classes of RNAs are all formed by cotranscriptional cleavage, but each one has a distinct mechanism for 3Ј-end formation (for reviews, see references 29 and 32). In poly(A) ϩ and histone pre-mRNAs there are conserved upstream and downstream sequences that flank the cleavage site; factors bind to these sites and then recruit additional factors that initiate cleavage (53). In the case of poly(A) ϩ pre-mRNA, the upstream element is the canonical AAUAAA polyadenylation signal (PAS) and the downstream sequence is the G/Urich downstream element (DSE). Recognition of the PAS is carried out by the cleavage and polyadenylation specificity complex (CPSF) component CPSF160 via its RNA recognition motifs (RRM) (36), whereas the DSE is bound by the RRM of the cleavage stimulation factor (CstF) component CstF64 (28). Subsequent to this recognition event is recruitment of additional factors that activate the endonucleolytic cleavage between the PAS and the DSE.Histone pre-mRNA contains a distinct set of flanking elements. Upstream of the cleavage site is a conserved stem-loop structure (SL) and downstream a purine-rich element called the ...

Metazoan replication-dependent histone mRNAs are not polyadenylated and instead end in a conserved stem loop that is the cis element responsible for coordinate posttranscriptional regulation of these mRNAs. Using biochemical approaches, only a limited number of factors required for cleavage of histone pre-mRNA have been identified. We therefore performed a genome-wide RNA interference screen in Drosophila cells using a GFP reporter that is expressed only when histone pre-mRNA processing is disrupted. Four of the 24 genes identified encode proteins also necessary for cleavage/polyadenylation, indicating mechanistic conservation in formation of different mRNA 3' ends. We also unexpectedly identified the histone variants H2Av and H3.3A/B. In H2Av mutant cells, U7 snRNP remains active but fails to accumulate at the histone locus, suggesting there is a regulatory pathway that coordinates the production of variant and canonical histones that acts via localization of essential histone pre-mRNA processing factors.

Metazoan replication-dependent histone mRNAs are not polyadenylated, and instead terminate in a conserved stem-loop structure generated by an endonucleolytic cleavage involving the U7 snRNP, which interacts with histone pre-mRNAs through base-pairing between U7 snRNA and a purine-rich sequence in the pre-mRNA located downstream of the cleavage site. Here we generate null mutations of the single Drosophila U7 gene and demonstrate that U7 snRNA is required in vivo for processing all replication-associated histone pre-mRNAs. Mutation of U7 results in the production of poly A + histone mRNA in both proliferating and endocycling cells because of read-through to cryptic polyadenylation sites found downstream of each Drosophila histone gene. A similar molecular phenotype also results from mutation of Slbp, which encodes the protein that binds the histone mRNA 3¢ stem-loop. U7 null mutants develop into sterile males and females, and these females display defects during oogenesis similar to germ line clones of Slbp null cells. In contrast to U7 mutants, Slbp null mutations cause lethality. This may reflect a later onset of the histone pre-mRNA processing defect in U7 mutants compared to Slbp mutants, due to maternal stores of U7 snRNA. A double mutant combination of a viable, hypomorphic Slbp allele and a viable U7 null allele is lethal, and these double mutants express polyadenylated histone mRNAs earlier in development than either single mutant. These data suggest that SLBP and U7 snRNP cooperate in the production of histone mRNA in vivo, and that disruption of histone pre-mRNA processing is detrimental to development.