FLASH is required for the endonucleolytic cleavage of histone pre-mRNAs but is dispensable for the 5' exonucleolytic degradation of the downstream cleavage product

3'-end cleavage of histone pre-mRNAs is catalyzed by CPSF-73 and requires the interaction of two U7 snRNP-associated proteins, FLASH and Lsm11. Here, by using scanning mutagenesis we identify critical residues in human FLASH and Lsm11 that are involved in the interaction between these two proteins. We also demonstrate that mutations in the region of FLASH located between amino acids 50 and 99 do not affect binding of Lsm11. Interestingly, these mutations convert FLASH into an inhibitory protein that reduces in vitro processing efficiency of highly active nuclear extracts. Our results suggest that this region in FLASH in conjunction with Lsm11 is involved in recruiting a yet-unknown processing factor(s) to histone pre-mRNA. Following endonucleolytic cleavage of histone pre-mRNA, the downstream cleavage product (DCP) is degraded by the 5'-3' exonuclease activity of CPSF-73, which also depends on Lsm11. Strikingly, while cleavage of histone pre-mRNA is stimulated by FLASH and inhibited by both dominant negative mutants of FLASH and anti-FLASH antibodies, the 5'-3' degradation of the DCP is not affected. Thus, the recruitment of FLASH to the processing complex plays a critical role in activating the endonuclease mode of CPSF-73 but is dispensable for its 5'-3' exonuclease activity. These results suggest that CPSF-73, the catalytic component in both reactions, can be recruited to histone pre-mRNA largely in a manner independent of FLASH, possibly by a separate domain in Lsm11.     

A 5'-3' exonuclease activity involved in forming the 3' products of histone pre-mRNA processing in vitro.

Histone RNA 3' processing in vitro produces one or more 5' cleavage products corresponding to the mature histone mRNA 3' end, and a group of 3' cleavage products whose 5' ends are mostly located several nucleotides downstream of the mRNA 3' end. The formation of these 3' products is coupled to the formation of 5' products and dependent on the U7 snRNP and a heat-labile processing factor. These short 3' products therefore are a true and general feature of the processing reaction. Identical 3' products are also formed from a model RNA containing all spacer nucleotides downstream of the mature mRNA 3' end, but no sequences from the mature mRNA. Again, this reaction is dependent on both the U7 snRNP and a heat-labile factor. Unlike the processing with a full-length histone pre-mRNA, this reaction produces only 3' but no 5' fragments. In addition, product formation is inhibited by addition of cap structures at the model RNA 5' end, indicating that product formation occurs by 5'-3' exonucleolytic degradation. This degradation of a model 3' product by a 5'-3' exonuclease suggests a mechanism for the release of the U7 snRNP after processing by shortening the cut-off histone spacer sequences base paired to U7 RNA.     

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

Animal replication-dependent histone pre-mRNAs are processed at the 3′ end by endonucleolytic cleavage that is not followed by polyadenylation. The cleavage reaction is catalyzed by CPSF73 and depends on the U7 snRNP and its integral component, Lsm11. A critical role is also played by the 220-kDa protein FLASH, which interacts with Lsm11. Here we demonstrate that the N-terminal regions of these two proteins form a platform that tightly interacts with a unique combination of polyadenylation factors: symplekin, CstF64, and all CPSF subunits, including the endonuclease CPSF73. The interaction is inhibited by alterations in each component of the FLASH/Lsm11 complex, including point mutations in FLASH that are detrimental for processing. The same polyadenylation factors are associated with the endogenous U7 snRNP and are recruited in a U7-dependent manner to histone pre-mRNA. Collectively, our studies identify the molecular mechanism that recruits the CPSF73 endonuclease to histone pre-mRNAs, reveal an unexpected complexity of the U7 snRNP, and suggest that in animal cells polyadenylation factors assemble into two alternative complexes—one specifically crafted to generate polyadenylated mRNAs and the other to generate nonpolyadenylated histone mRNAs that end with the stem-loop.     

Binding of human SLBP on the 3'-UTR of histone precursor H4-12 mRNA induces structural rearrangements that enable U7 snRNA anchoring

In metazoans, cell-cycle-dependent histones are produced from poly(A)-lacking mRNAs. The 3′ end of histone mRNAs is formed by an endonucleolytic cleavage of longer precursors between a conserved stem–loop structure and a purine-rich histone downstream element (HDE). The cleavage requires at least two trans-acting factors: the stem–loop binding protein (SLBP), which binds to the stem–loop and the U7 snRNP, which anchors to histone pre-mRNAs by annealing to the HDE. Using RNA structure-probing techniques, we determined the secondary structure of the 3′-untranslated region (3′-UTR) of mouse histone pre-mRNAs H4–12, H1t and H2a–614. Surprisingly, the HDE is embedded in hairpin structures and is therefore not easily accessible for U7 snRNP anchoring. Probing of the 3′-UTR in complex with SLBP revealed structural rearrangements leading to an overall opening of the structure especially at the level of the HDE. Electrophoretic mobility shift assays demonstrated that the SLBP-induced opening of HDE actually facilitates U7 snRNA anchoring on the histone H4–12 pre-mRNAs 3′ end. These results suggest that initial binding of the SLBP functions in making the HDE more accessible for U7 snRNA anchoring.     

U7 snRNP-specific Lsm11 protein: dual binding contacts with the 100 kDa zinc finger processing factor (ZFP100) and a ZFP100-independent function in histone RNA 3' end processing

The 3′ cleavage generating non-polyadenylated animal histone mRNAs depends on the base pairing between U7 snRNA and a conserved histone pre-mRNA downstream element. This interaction is enhanced by a 100 kDa zinc finger protein (ZFP100) that forms a bridge between an RNA hairpin element upstream of the processing site and the U7 small nuclear ribonucleoprotein (snRNP). The N-terminus of Lsm11, a U7-specific Sm-like protein, was shown to be crucial for histone RNA processing and to bind ZFP100. By further analysing these two functions of Lsm11, we find that Lsm11 and ZFP100 can undergo two interactions, i.e. between the Lsm11 N-terminus and the zinc finger repeats of ZFP100, and between the N-terminus of ZFP100 and the Sm domain of Lsm11, respectively. Both interactions are not specific for the two proteins in vitro, but the second interaction is sufficient for a specific recognition of the U7 snRNP by ZFP100 in cell extracts. Furthermore, clustered point mutations in three phylogenetically conserved regions of the Lsm11 N-terminus impair or abolish histone RNA processing. As these mutations have no effect on the two interactions with ZFP100, these protein regions must play other roles in histone RNA processing, e.g. by contacting the pre-mRNA or additional processing factors.     

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

3′-End cleavage of animal replication-dependent histone pre-mRNAs is controlled by the U7 snRNP. Lsm11, the largest component of the U7-specific Sm ring, interacts with FLASH, and in mammalian nuclear extracts these two proteins form a platform that recruits the CPSF73 endonuclease and other polyadenylation factors to the U7 snRNP. FLASH is limiting, and the majority of the U7 snRNP in mammalian extracts exists as a core particle consisting of the U7 snRNA and the Sm ring. Here, we purified the U7 snRNP from Drosophila nuclear extracts and characterized its composition by mass spectrometry. In contrast to the mammalian U7 snRNP, a significant fraction of the Drosophila U7 snRNP contains endogenous FLASH and at least six subunits of the polyadenylation machinery: symplekin, CPSF73, CPSF100, CPSF160, WDR33, and CstF64. The same composite U7 snRNP is recruited to histone pre-mRNA for 3′-end processing. We identified a motif in Drosophila FLASH that is essential for the recruitment of the polyadenylation complex to the U7 snRNP and analyzed the role of other factors, including SLBP and Ars2, in 3′-end processing of Drosophila histone pre-mRNAs. SLBP that binds the upstream stem–loop structure likely recruits a yet-unidentified essential component(s) to the processing machinery. In contrast, Ars2, a protein previously shown to interact with FLASH in mammalian cells, is dispensable for processing in Drosophila. Our studies also demonstrate that Drosophila symplekin and three factors involved in cleavage and polyadenylation—CPSF, CstF, and CF I_m—are present in Drosophila nuclear extracts in a stable supercomplex.     

The 68?kDa subunit of mammalian cleavage factor I interacts with the U7 small nuclear ribonucleoprotein and participates in 3′-end processing of animal histone mRNAs

Metazoan replication-dependent histone pre-mRNAs undergo a unique 3′-cleavage reaction which does not result in mRNA polyadenylation. Although the cleavage site is defined by histone-specific factors (hairpin binding protein, a 100-kDa zinc-finger protein and the U7 snRNP), a large complex consisting of cleavage/polyadenylation specificity factor, two subunits of cleavage stimulation factor and symplekin acts as the effector of RNA cleavage. Here, we report that yet another protein involved in cleavage/polyadenylation, mammalian cleavage factor I 68-kDa subunit (CF I_m68), participates in histone RNA 3′-end processing. CF I_m68 was found in a highly purified U7 snRNP preparation. Its interaction with the U7 snRNP depends on the N-terminus of the U7 snRNP protein Lsm11, known to be important for histone RNA processing. In vivo, both depletion and overexpression of CF I_m68 cause significant decreases in processing efficiency. In vitro 3′-end processing is slightly stimulated by the addition of low amounts of CF I_m68, but inhibited by high amounts or by anti-CF I_m68 antibody. Finally, immunoprecipitation of CF I_m68 results in a strong enrichment of histone pre-mRNAs. In contrast, the small CF I_m subunit, CF I_m25, does not appear to be involved in histone RNA processing.     

The site of 3'' end formation of histone messenger RNA is a fixed distance from the downstream element recognized by the U7 snRNP.

Two conserved elements direct the 3' end processing of histone messenger RNA: a stem-loop structure immediately upstream of the site of cleavage and the histone downstream element (HDE), located 12-19 nucleotides downstream of the stem-loop in the premessenger RNA. We studied the role of these two elements by systematically inserting up to 10 C residues between them in the mouse H2A-614 histone pre-mRNA. 3' End mapping of RNAs processed in vitro demonstrated that as the HDE is move downstream, the site of cleavage correspondingly moves 3'. In addition, the efficiency of processing declines. In the wild-type substrate, cleavage occurs 3' of an A residue; modest increases in the efficiency of processing of the insertion mutants were observed when an A residue was placed at the new cleavage site. The results of psoralen cross-linking studies and immunoprecipitations using anti-trimethylguanosine antibodies indicated that the decreased processing efficiency of the insertion mutants is not due to impaired binding of the U7 small nuclear ribonucleoprotein (snRNP). We conclude that the mammalian U7 snRNP acts as a molecular ruler, targeting enzymatic components of cleave histone pre-mRNAs a fixed distance from its binding site, the HDE.     

The Pre-mRNA 5′ Cap Determines Whether U6 Small Nuclear RNA Succeeds U1 Small Nuclear Ribonucleoprotein Particle at 5′ Splice Sites

Efficient splicing of the 5′-most intron of pre-mRNA requires a 5′ m⁷G(5′)ppp(5′)N cap, which has been implicated in U1 snRNP binding to 5′ splice sites. We demonstrate that the cap alters the kinetic profile of U1 snRNP binding, but its major effect is on U6 snRNA binding. With two alternative wild-type splice sites in an adenovirus pre-mRNA, the cap selectively alters U1 snRNA binding at the site to which cap-independent U1 snRNP binding is stronger and that is used predominantly in splicing; with two consensus sites, the cap acts on both, even though one is substantially preferred for splicing. However, the most striking quantitative effect of the 5′ cap is neither on U1 snRNP binding nor on the assembly of large complexes but on the replacement of U1 snRNP by U6 snRNA at the 5′ splice site. Inhibition of splicing by a cap analogue is correlated with the loss of U6 interactions at the 5′ splice site and not with any loss of U1 snRNP binding.     

U7 snRNA mutations in Drosophila block histone pre-mRNA processing and disrupt oogenesis

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.     

Formation of the 3' end of histone mRNA

All metazoan messenger RNAs, with the exception of the replication-dependent histone mRNAs, terminate at the 3' end with a poly(A) tail. Replication-dependent histone mRNAs end instead in a conserved 26-nucleotide sequence that contains a 16-nucleotide stem-loop. Formation of the 3' end of histone mRNA occurs by endonucleolytic cleavage of pre-mRNA releasing the mature mRNA from the chromatin template. Cleavage requires several trans-acting factors, including a protein, the stem-loop binding protein (SLBP), which binds the 26-nucleotide sequence; and a small nuclear RNP, U7 snRNP. There are probably additional factors also required for cleavage. One of the functions of the SLBP is to stabilize binding of the U7 snRNP to the histone pre-mRNA. In the nucleus, both U7 snRNP and SLBP are present in coiled bodies, structures that are associated with histone genes and may play a direct role in histone pre-mRNA processing in vivo. One of the major regulatory events in the cell cycle is regulation of histone pre-mRNA processing, which is at least partially mediated by cell-cycle regulation of the levels of the SLBP protein.     

Differences and similarities between Drosophila and mammalian 3' end processing of histone pre-mRNAs

We used nuclear extracts from Drosophila Kc cells to characterize 3' end processing of Drosophila histone pre-mRNAs. Drosophila SLBP plays a critical role in recruiting the U 7 snRNP to the pre-mRNA and is essential for processing all five Drosophila histone pre-mRNAs. The Drosophila processing machinery strongly prefers cleavage after a fourth nucleotide following the stem-loop and favors an adenosine over pyrimidines in this position. Increasing the distance between the stem-loop and the HDE does not result in a corresponding shift of the cleavage site, suggesting that in Drosophila processing the U 7 snRNP does not function as a molecular ruler. Instead, SLBP directs the cleavage site close to the stem-loop. The upstream cleavage product generated in Drosophila nuclear extracts contains a 3' OH, and the downstream cleavage product is degraded by a nuclease dependent on the U 7 snRNP, suggesting that the cleavage factor has been conserved between Drosophila and mammalian processing. A 2'O-methyl oligonucleotide complementary to the first 17 nt of the Drosophila U 7 snRNA was not able to deplete the U 7 snRNP from Drosophila nuclear extracts, suggesting that the 5' end of the Drosophila U 7 snRNA is inaccessible. This oligonucleotide selectively inhibited processing of only two Drosophila pre-mRNAs and had no effect on processing of the other three pre-mRNAs. Together, these studies demonstrate that although Drosophila and mammalian histone pre-mRNA processing share common features, there are also significant differences, likely reflecting divergence in the mechanism of 3' end processing between vertebrates and invertebrates.