Loss of the Histone Pre-mRNA Processing Factor Stem-Loop Binding Protein in Drosophila Causes Genomic Instability and Impaired Cellular Proliferation

BACKGROUND:Metazoan replication-dependent histone mRNAs terminate in a conserved stem-loop structure rather than a polyA tail. Formation of this unique mRNA 3' end requires Stem-loop Binding Protein (SLBP), which directly binds histone pre-mRNA and stimulates 3' end processing. The 3' end stem-loop is necessary for all aspects of histone mRNA metabolism, including replication coupling, but its importance to organism fitness and genome maintenance in vivo have not been characterized. METHODOLOGY/PRINCIPAL FINDINGS:In Drosophila, disruption of the Slbp gene prevents normal histone pre-mRNA processing and causes histone pre-mRNAs to utilize the canonical 3' end processing pathway, resulting in polyadenylated histone mRNAs that are no longer properly regulated. Here we show that Slbp mutants display genomic instability, including loss of heterozygosity (LOH), increased presence of chromosome breaks, tetraploidy, and changes in position effect variegation (PEV). During imaginal disc growth, Slbp mutant cells show defects in S phase and proliferate more slowly than control cells. CONCLUSIONS/SIGNIFICANCE:These data are consistent with a model in which changing the 3' end of histone mRNA disrupts normal replication-coupled histone mRNA biosynthesis and alters chromatin assembly, resulting in genomic instability, inhibition of cell proliferation, and impaired development

A Genome-wide RNA Interference Screen Reveals that Variant Histones Are Necessary for Replication-Dependent Histone Pre-mRNA Processing

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

A Sequence in the Drosophila H3-H4 Promoter Triggers Histone Locus Body Assembly and Biosynthesis of Replication-Coupled Histone mRNAs

Compartmentalization of RNA biosynthetic factors into nuclear bodies (NBs) is a ubiquitous feature of eukaryotic cells. How NBs initially assemble and ultimately affect gene expression remains unresolved. The histone locus body (HLB) contains factors necessary for replication-coupled histone mRNA transcription and processing and associates with histone gene clusters. Using a transgenic assay for ectopic Drosophila HLB assembly, we show that a sequence located between, and transcription from, the divergently transcribed H3-H4 genes nucleates HLB formation and activates other histone genes in the histone gene cluster. In the absence of transcription from the H3-H4 promoter, “proto-HLBs”, containing only a subset of HLB components, form and the adjacent histone H2a-H2b genes are not expressed. Proto-HLBs also transiently form in mutant embryos with the histone locus deleted. We conclude that HLB assembly occurs through a stepwise process involving stochastic interactions of individual components that localize to a specific sequence in the H3-H4 promoter

<i>Slbp</i> mutants have no detectable global change in histone protein levels.

<p>Protein lysates from <i>wt, Slbp¹⁵, Slbp¹⁰</i>, and <i>H2aV⁸¹⁰</i> mutants were obtained from whole 3^rd instar larvae and probed with antibodies to A) H2b and H3, and B) H3K4-me2 and H3K9-me2. β-tubulin was used as a loading control for both panels.</p

Mean Percentage of Mitotic Nuclei Per Brain in Each Class.

<p>For each genotype, the percentage of nuclei containing chromosomal breaks and tetraploidy was calculated for 6 individual brains and mean percentages determined. 23–132 mitotic nuclei per individual brain were analyzed. Four classes of abnormal karyotypes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008168#pone-0008168-g002" target="_blank">Figure 2</a>): I) Normal karyotype with no breaks. II) Diploid nuclei containing breaks. II + IV) All nuclei containing breaks. III) Tetraploid nuclei without breaks. III + IV) Total tetraploid nuclei including those with breaks. IV) Tetraploid nuclei with breaks.</p

LOH is significantly increased in <i>Slbp</i> mutants.

<p>Wings from wt, <i>Slbp¹⁰</i> mutant flies heterozygous for a fourth chromosome translocation containing <i>y⁺</i> were mounted and the first twenty bristles of the anterior wing margin were used for analysis. A) Example of the data used for analysis. Yellow bristles indicate an LOH event (black arrows). B) For each <i>Slbp¹⁰</i> and<i> + </i>class, the frequencies of yellow bristles per wing were compared to heterozygous controls derived from a common culture. Frequency of yellow bristles was tabulated on a per wing basis. Data are expressed as a percentage of heterozygous controls, so that genotypes can be compared across experiments. 21-45 wings were analyzed per class. P-values are indicated as follows: * p<0.05, **p<0.01, ***p<0.001.</p

<i>Slbp</i> mutants have increased DNA damage and exhibit tetraploid nuclei.

<p>Larval neuroblast karyotypes were obtained for <i>Slbp¹⁵</i> and <i>w¹¹¹⁸</i> 3^rd instar larvae. Each karyotype was assigned to one of four categories: I) Normal karyotype with no chromosomal breaks II) Normal karyotype with at least one chromosomal break (arrow), III) Tetraploid with no breaks, IV) Tetraploid with at least one break (arrows). White arrows indicate examples of chromosomal breaks.</p

<i>Slbp</i> mutants are modifiers of PEV.

<p>Eyes were analyzed for extent of variegation in flies carrying one copy of the <i>w^m4</i> inversion with one of the following genotypes: <i>Slbp¹⁵/Slbp¹⁰, Slbp¹⁵/+,</i> and <i>wt</i>. A) Representative eyes from females of each genotype. B) Quantitative assay for PEV. Eye pigment was quantified by measuring absorbance at 480λ for 30 fly heads per sample. C) Absorbance values of samples from allelic combinations of <i>Slbp</i> mutations. For each genotype, n = 10, except when comparing <i>Slbp¹⁵/Slbp¹⁰</i> and <i>Slbp¹⁵/+</i>, for which n = 3. Each pair of bars represents a single experiment. P-values are indicated as follows: * p<0.05, **p<0.01, ***p<0.001. Error bars indicate SEM.</p