Interaction between FLASH and Lsm11 is essential for histone pre-mRNA processing in vivo in Drosophila

Metazoan replication-dependent histone mRNAs are the only nonpolyadenylated cellular mRNAs. Formation of the histone mRNA 3′ end requires the U7 snRNP, which contains Lsm10 and Lsm11, and FLASH, a processing factor that binds Lsm11. Here, we identify sequences in Drosophila FLASH (dFLASH) that bind Drosophila Lsm11 (dLsm11), allow localization of dFLASH to the nucleus and histone locus body (HLB), and participate in histone pre-mRNA processing in vivo. Amino acids 105–154 of dFLASH bind to amino acids 1–78 of dLsm11. A two-amino acid mutation of dLsm11 that prevents dFLASH binding but does not affect localization of U7 snRNP to the HLB cannot rescue the lethality or histone pre-mRNA processing defects resulting from an Lsm11 null mutation. The last 45 amino acids of FLASH are required for efficient localization to the HLB in Drosophila cultured cells. Removing the first 64 amino acids of FLASH has no effect on processing in vivo. Removal of 13 additional amino acids of dFLASH results in a dominant negative protein that binds Lsm11 but inhibits processing of histone pre-mRNA in vivo. Inhibition requires the Lsm11 binding site, suggesting that the mutant dFLASH protein sequesters the U7 snRNP in an inactive complex and that residues between 64 and 77 of dFLASH interact with a factor required for processing. Together, these studies demonstrate that direct interaction between dFLASH and dLsm11 is essential for histone pre-mRNA processing in vivo and for proper development and viability in flies

Differentially expressed alternatively spliced genes in Malignant Pleural Mesothelioma identified using massively parallel transcriptome sequencing

<p>Abstract</p> <p>Background</p> <p>Analyses of Expressed Sequence Tags (ESTs) databases suggest that most human genes have multiple alternative splice variants. The alternative splicing of pre-mRNA is tightly regulated during development and in different tissue types. Changes in splicing patterns have been described in disease states. Recently, we used whole-transcriptome shotgun pryrosequencing to characterize 4 malignant pleural mesothelioma (MPM) tumors, 1 lung adenocarcinoma and 1 normal lung. We hypothesized that alternative splicing profiles might be detected in the sequencing data for the expressed genes in these samples.</p> <p>Methods</p> <p>We developed a software pipeline to map the transcriptome read sequences of the 4 MPM samples and 1 normal lung sample onto known exon junction sequences in the comprehensive AceView database of expressed sequences and to count how many reads map to each junction. 13,274,187 transcriptome reads generated by the Roche/454 sequencing platform for 5 samples were compared with 151,486 exon junctions from the AceView database. The exon junction expression index (EJEI) was calculated for each exon junction in each sample to measure the differential expression of alternative splicing events. Top ten exon junctions with the largest EJEI difference between the 4 mesothelioma and the normal lung sample were then examined for differential expression using Quantitative Real Time PCR (qRT-PCR) in the 5 sequenced samples. Two of the differentially expressed exon junctions (ACTG2.aAug05 and CDK4.aAug05) were further examined with qRT-PCR in additional 18 MPM and 18 normal lung specimens.</p> <p>Results</p> <p>We found 70,953 exon junctions covered by at least one sequence read in at least one of the 5 samples. All 10 identified most differentially expressed exon junctions were validated as present by RT-PCR, and 8 were differentially expressed exactly as predicted by the sequence analysis. The differential expression of the AceView exon junctions for the ACTG2 and CDK4 genes were also observed to be statistically significant in an additional 18 MPM and 18 normal lung samples examined using qRT-PCR. The differential expression of these two junctions was shown to successfully classify these mesothelioma and normal lung specimens with high sensitivity (89% and 78%, respectively).</p> <p>Conclusion</p> <p>Whole-transcriptome shotgun sequencing, combined with a downstream bioinformatics pipeline, provides powerful tools for the identification of differentially expressed exon junctions resulting from alternative splice variants. The alternatively spliced genes discovered in the study could serve as useful diagnostic markers as well as potential therapeutic targets for MPM.</p

Interaction between FLASH and Lsm11 is essential for histone pre-mRNA processing in vivo in Drosophila

Metazoan replication-dependent histone mRNAs are the only nonpolyadenylated cellular mRNAs. Formation of the histone mRNA 3′ end requires the U7 snRNP, which contains Lsm10 and Lsm11, and FLASH, a processing factor that binds Lsm11. Here, we identify sequences in Drosophila FLASH (dFLASH) that bind Drosophila Lsm11 (dLsm11), allow localization of dFLASH to the nucleus and histone locus body (HLB), and participate in histone pre-mRNA processing in vivo. Amino acids 105–154 of dFLASH bind to amino acids 1–78 of dLsm11. A two-amino acid mutation of dLsm11 that prevents dFLASH binding but does not affect localization of U7 snRNP to the HLB cannot rescue the lethality or histone pre-mRNA processing defects resulting from an Lsm11 null mutation. The last 45 amino acids of FLASH are required for efficient localization to the HLB in Drosophila cultured cells. Removing the first 64 amino acids of FLASH has no effect on processing in vivo. Removal of 13 additional amino acids of dFLASH results in a dominant negative protein that binds Lsm11 but inhibits processing of histone pre-mRNA in vivo. Inhibition requires the Lsm11 binding site, suggesting that the mutant dFLASH protein sequesters the U7 snRNP in an inactive complex and that residues between 64 and 77 of dFLASH interact with a factor required for processing. Together, these studies demonstrate that direct interaction between dFLASH and dLsm11 is essential for histone pre-mRNA processing in vivo and for proper development and viability in flies