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

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 factor

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 Im68), participates in histone RNA 3′-end processing. CF Im68 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 Im68 cause significant decreases in processing efficiency. In vitro 3′-end processing is slightly stimulated by the addition of low amounts of CF Im68, but inhibited by high amounts or by anti-CF Im68 antibody. Finally, immunoprecipitation of CF Im68 results in a strong enrichment of histone pre-mRNAs. In contrast, the small CF Im subunit, CF Im25, does not appear to be involved in histone RNA processin

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 Im68), participates in histone RNA 3′-end processing. CF Im68 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 Im68 cause significant decreases in processing efficiency. In vitro 3′-end processing is slightly stimulated by the addition of low amounts of CF Im68, but inhibited by high amounts or by anti-CF Im68 antibody. Finally, immunoprecipitation of CF Im68 results in a strong enrichment of histone pre-mRNAs. In contrast, the small CF Im subunit, CF Im25, does not appear to be involved in histone RNA processing

Maf1, a New Player in the Regulation of Human RNA Polymerase III Transcription

BACKGROUND: Human RNA polymerase III (pol III) transcription is regulated by several factors, including the tumor suppressors P53 and Rb, and the proto-oncogene c-Myc. In yeast, which lacks these proteins, a central regulator of pol III transcription, called Maf1, has been described. Maf1 is required for repression of pol III transcription in response to several signal transduction pathways and is broadly conserved in eukaryotes. METHODOLOGY/PRINCIPAL FINDINGS: We show that human endogenous Maf1 can be co-immunoprecipitated with pol III and associates in vitro with two pol III subunits, the largest subunit RPC1 and the α-like subunit RPAC2. Maf1 represses pol III transcription in vitro and in vivo and is required for maximal pol III repression after exposure to MMS or rapamycin, treatments that both lead to Maf1 dephosphorylation. CONCLUSIONS/SIGNIFICANCE: These data suggest that Maf1 is a major regulator of pol III transcription in human cells

Evolutionary conservation of the U7 small nuclear ribonucleoprotein in Drosophila melanogaster

The U7 snRNP involved in histone RNA 3′ end processing is related to but biochemically distinct from spliceosomal snRNPs. In vertebrates, the Sm core structure assembling around the noncanonical Sm-binding sequence of U7 snRNA contains only five of the seven standard Sm proteins. The missing Sm D1 and D2 subunits are replaced by U7-specific Sm-like proteins Lsm10 and Lsm11, at least the latter of which is important for histone RNA processing. So far, it was unknown if this special U7 snRNP composition is conserved in invertebrates. Here we describe several putative invertebrate Lsm10 and Lsm11 orthologs that display low but clear sequence similarity to their vertebrate counterparts. Immunoprecipitation studies in Drosophila S2 cells indicate that the Drosophila Lsm10 and Lsm11 orthologs (dLsm10 and dLsm11) associate with each other and with Sm B, but not with Sm D1 and D2. Moreover, dLsm11 associates with the recently characterized Drosophila U7 snRNA and, indirectly, with histone H3 pre-mRNA. Furthermore, dLsm10 and dLsm11 can assemble into U7 snRNPs in mammalian cells. These experiments demonstrate a strong evolutionary conservation of the unique U7 snRNP composition, despite a high degree of primary sequence divergence of its constituents. Therefore, Drosophila appears to be a suitable system for further genetic studies of the cell biology of U7 snRNPs

The N-terminus of Lsm11 (GST-mLsm11) binds to the C2H2 zinc finger repeats of ZFP100

<p><b>Copyright information:</b></p><p>Taken from "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"</p><p>Nucleic Acids Research 2005;33(7):2106-2117.</p><p>Published online 11 Apr 2005</p><p>PMCID:PMC1075925.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Structure of ZFP100. Dark grey box labelled ‘K’, KRAB domain; light grey boxes, C2H2 zinc finger repeats; imperfect repeats are delineated by stippled lines. () GST pull-down assays with ZFP, ZFP and ZFP encoded by subcloned NcoI and NcoI/XhoI restriction fragments as indicated in (A). () GST pull-down assays performed with ZFP100 truncations obtained by PCR (see Materials and Methods). The numbers indicate the ranges of amino acids of FL ZFP100 that are present in the various truncations. () GST pull-down assay performed with the C2H2 zinc finger protein Kid-1, a renal transcription factor from rat that is not related to histone RNA processing. All templates were linearized and subjected to coupled transcription/translation in the presence of [S]methionine. The translation products were incubated with GST-mLsm11 or GST (negative control) immobilized on glutathione sepharose beads. The beads were washed, and the bound material was analysed by SDS–PAGE and autoradiography. Input, 1/10 the amount used in the binding assays was analysed directly. Note that only ZFP encoding the N-terminus of ZFP100 lacking zinc finger repeats but containing the KRAB domain does not bind to GST-mLsm11, whereas all fragments encoding zinc finger repeats bind efficiently

Conserved amino acid sequences in the N-terminus of Lsm11 are not important for binding to ZFP100

<p><b>Copyright information:</b></p><p>Taken from "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"</p><p>Nucleic Acids Research 2005;33(7):2106-2117.</p><p>Published online 11 Apr 2005</p><p>PMCID:PMC1075925.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Mutagenesis of four phylogenetically conserved amino acid sequences in the N-terminus of Lsm11 () (see our Lsm11 database at ). Amino acids that were mutated to alanines in the murine Lsm11 cDNA are underlined and the amino acid position of the first one is indicated. The mutants were named according to the original sequence (i.e. PLL, YES…). () The S-labelled Lsm11 (FL) and its mutants were generated in reticulocyte lysate and incubated with equal amounts of GST (data not shown, but no signals were obtained) or GST-ZFP100 N-terminal fragment (GST-ZFP). For a description of the Lsm11 FL and ΔN140, see . () The S-labelled ZFP fragments ZFP (upper panel) and ZFP (lower panel) were generated in reticulocyte lysate and incubated with beads loaded with GST (lane 2) or GST-tagged Lsm11 amino acids 1–136 (lanes 3–6) or 1–157 (lanes 7,8) that contained either the wt sequence or the indicated mutations

Model depicting the interactions between Lsm11 and ZFP100 characterized in this paper

<p><b>Copyright information:</b></p><p>Taken from "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"</p><p>Nucleic Acids Research 2005;33(7):2106-2117.</p><p>Published online 11 Apr 2005</p><p>PMCID:PMC1075925.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> To illustrate the interactions, Lsm11 (bottom) has been drawn in the opposite orientation relative to ZFP100 (top). An interaction between the zinc finger repeats 2–8 of ZFP100 and the HBP–histone RNA hairpin complex was previously characterized by Dominski, Marzluff and co-workers (). The three conserved motifs in the N-terminus of Lsm11, which are important for histone RNA processing (PLL, PER and MPL), are shown in red, the fourth motif (YES) in grey