The Drosophila U7 snRNP proteins Lsm10 and Lsm11 are required for histone pre-mRNA processing and play an essential role in development

Abstract

Metazoan replication-dependent histone mRNAs are not polyadenylated, and instead terminate in a conserved stem-loop structure generated by an endonucleolytic cleavage of the pre-mRNA involving U7 snRNP. U7 snRNP contains two like-Sm proteins, Lsm10 and Lsm11, which replace SmD1 and SmD2 in the canonical heptameric Sm protein ring that binds spliceosomal snRNAs. Here we show that mutations in either the Drosophila Lsm10 or the Lsm11 gene disrupt normal histone pre-mRNA processing, resulting in production of poly(A)+ histone mRNA as a result of transcriptional read-through to cryptic polyadenylation sites present downstream from each histone gene. This molecular phenotype is indistinguishable from that which we previously described for mutations in U7 snRNA. Lsm10 protein fails to accumulate in Lsm11 mutants, suggesting that a pool of Lsm10-Lsm11 dimers provides precursors for U7 snRNP assembly. Unexpectedly, U7 snRNA was detected in Lsm11 and Lsm1 mutants and could be precipitated with anti-trimethylguanosine antibodies, suggesting that it assembles into a snRNP particle in the absence of Lsm10 and Lsm11. However, this U7 snRNA could not be detected at the histone locus body, suggesting that Lsm10 and Lsm11 are necessary for U7 snRNP localization. In contrast to U7 snRNA null mutants, which are viable, Lsm10 and Lsm11 mutants do not survive to adulthood. Because we cannot detect differences in the histone mRNA phenotype between Lsm10 or Lsm11 and U7 mutants, we propose that the different terminal developmental phenotypes result from the participation of Lsm10 and Lsm11 in an essential function that is distinct from histone pre-mRNA processing and that is independent of U7 snRNA.

FIGURE 1.

Identification of Lsm10 and Lsm11 mutations. (A) Schematic diagram of the Lsm11 and Lsm10 genes. The black bars represent the coding sequence and the gray bars represent the 5′ and 3′ UTRs. Note there are no introns in these two genes. The position of the Sm1 and Sm2 domains are marked with black bars above the coding sequence. The positions of the pBac insertions are indicated by black triangles. The positions of the three Lsm10 EMS alleles are indicated below the gene. (B–D) Protein extracts of brain and salivary gland tissue from third instar larvae or adult flies (D) of the indicated genotypes were probed with anti-Lsm11 or anti Lsm10-antibodies by Western blotting. w¹¹¹⁸ was used as a normal control here and in subsequent figures (+). α-Tubulin is used as a loading control.

FIGURE 2.

Lsm10 and Lsm11 mutants fail to properly process histone pre-mRNA and are necessary for development. (A) Total RNA isolated from whole third instar larvae of the indicated genotypes was subjected to Northern analysis with a ³²P-labeled H3 probe. Note that the severity of the misprocessed H3 phenotype is similar in U7²⁰, Lsm10^G40E, and Lsm11^c02047 mutants, while the terminal developmental phenotype is different: viable for U7²⁰ and not viable for the Lsm10^G40E and Lsm11^c02047 null mutants. The Lsm10^f06616 hypomorph is viable. (B) Protein extracts prepared from embryos of the indicated genotypes were probed with anti-V5 antibodies by Western blotting. P[Lsm11⁺] is a transgene expressing a V5-Lsm11 with the endogenous Lsm11 promoter. “11” Refers to the homozygous Lsm11^c02047 genotype. Lane 3 contains protein from a nontransgenic control. Note that in a wild-type Lsm11 background there is very little accumulation of V5-Lsm11 protein. α–Tubulin is used as a loading control. (C) Protein extracts isolated from whole third instar larvae of the indicated genotypes were subjected to immunoprecipitation then Western blot analysis with anti-V5 antibody. Lane 2 contains protein from nontransgenic control. (D,E) RNA isolated from whole third instar larvae of the indicated genotypes was subjected to Northern analysis with ³²P-labeled H3 probe. “10” Refers to the Lsm10^G40E/Df mutant genotype. Note that there is very little misprocessed H3 in both Lsm11^c02047, P[Lsm11⁺] and Lsm10^G40E/Df, P[Lsm10⁺] genotypes. (F) Protein extracts prepared from whole third instar larvae of the indicated genotypes were probed with anti-Lsm10 antibodies. Note that the lane 1 genotype contains a single copy of P[Lsm10⁺], accounting for the reduction in Lsm10 accumulation relative to wild type (+). All other P[Lsm] transgenes are present in two copies.

FIGURE 3.

Lsm11 mutant lethality is independent of histone mRNA misprocessing. (A–C) Total RNA was extracted from animals at different stages of development of the indicated genotypes and subjected to Northern analysis with a ³²P-labeled probe to H2B (B,C) or H3 (A). Note that the misprocessed histone mRNA is first detectable in small amounts at the second larval instar stage and that wild-type histone mRNA is absent in the third larval instar stage.

FIGURE 4.

Quantitative analysis of histone mRNA levels between U7 and Lsm11 mutants. (A,B) Total RNA was extracted from first instar larvae of the indicated genotypes and different quantities subjected to Northern analysis with a ³²P-labeled probe to H2B (A) or H3 (B). U1 is used as a loading control.

FIGURE 5.

Hypomorphic Lsm10 alleles are viable, with some fertility defects. (A) The average and standard deviation of the percent of hatched embryos for the indicated genotypes. Measurements were made on six collections of 100 eggs. (B) RNA isolated from 1–2-d-old adult females of the indicated genotypes was subjected to Northern analysis with a ³²P-labeled H3 probe. Note misprocessed H3 mRNA is detected in only two of the Lsm10 mutant genotypes, and that these hypomorphic mutants also contain wild-type, processed mRNA.

FIGURE 6.

U7 snRNA can form a snRNP particle in Lsm11 mutants. (A) U7 Northern analysis of RNA isolated from Lsm11^c02047/Df mutant (Lsm11) or w¹¹¹⁸ control (WT) embryos and first to third instar larvae. Note that in the Lsm11 mutant U7 snRNA is detected in third instar larvae when all the histone mRNA is misprocessed. A U1 probe was used as a loading control. U7 snRNA migrates as a doublet as described previously (Dominski et al. 2003). (B) Reverse transcriptase (RT)-PCR analysis of RNA extracted from anti-TMG immunoprecipitates of whole third instar larvae RNA samples of the indicated genotypes. (Lanes 1–6) 10% of total input RNA; (lanes 7–12) anti-TMG IP; and (lanes 13,14) mock IP negative control. (Top panel) U7 snRNA primer pair. Note that there is no U7 present in the U7^EY11305 mutant or in the control IP lane, but U7 is detected in both WT and Lsm11^c02047/Df TMG IP samples. (Middle panel) U1 snRNA primer pair. Note that U1 is present in all three TMG IP samples, but not in the IP control. (Bottom panel) Ribosomal protein 49 (rp49) primer pair. Note that rp49 mRNA is not precipitated by anti-TMG antibodies because the mRNA lacks a trimethylguanosine cap.

FIGURE 7.

The U7 snRNP formed in an Lsm11 mutant does not localize to the histone locus body. (A) Stage 5 Lsm11^c02047, P[V5-Lsm11⁺] homozygous embryos were stained with anti-Discs Large antibodies, to visualize cell boundaries, and anti-V5 antibodies (left panels, both red in merge). Anti-mouse secondary antibodies were used to simultaneously detect V5-Lsm11 and Discs Large. Embryos were also stained with DAPI (blue in merge). Note that V5-Lsm11 localizes to one or two nuclear foci just like endogenous Lsm11. Arrows indicate the same cell in (A) (20 μm scale bar) and (B) (10 μm scale bar). (C–F) Brains dissected from w¹¹¹⁸, U7^EY11305, Lsm10^G40E/Df, and Lsm11^c02047/Df third instar larvae were stained with MPM-2 (first column; green in merge), hybridized with a fluorescent probe recognizing U7 snRNA (second column; magenta in merge), anti-Lsm10 antibodies (third column; red in merge), and DAPI (blue in merge). Arrows indicate a histone locus body that contains MPM-2 antigen(s), U7, and Lsm10. (Insets) A higher magnification view. Arrowheads indicate a histone locus body lacking MPM-2 staining. This nucleus is likely not within S phase, and therefore lacks the Cyclin E/Cdk2 activity necessary to produce the MPM-2 epitope. Note that both U7 and Lsm10 are undetectable in histone locus bodies marked by MPM-2 staining in both Lsm10 (E) and Lsm11 (F) mutant brains. Bar, 10 μm (main panels) and bar, 5 μm (insets).