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Ssb1/2-IntS complexes maintain splicing integrity and genomic stability through regulating RNA Polymerase II mediated snRNA levels

ABSTRACT: Ssb1(NABP2/OBFC2A) and Ssb2 (NABP1/OBFC2B) are two closely related proteins that show functional redundancies supported by their existence in two separate subcomplexes with identical proteins. Ssb1 knockout causes perinatal lethality in mice, Ssb2 knockout does not associate with any observable phenotypes, however, double knockout of both Ssb1/2 causes embryonically lethality. Notably, conditional loss of both in adult mice leads to acute lethality due to bone marrow and gut failure. Here, we further demonstrate that the overlapping essential function of both Ssb1/2 is through their prime binding partner IntS3, a member of the multiprotein complex called the Integrator. The Integrator complex interacts with the largest subunit of RNA Polymerase II and is required for processing and termination of small nuclear RNAs (snRNAs). Here, we provide a firsthand evidence that the stability of the Integrator complex is compromised in mouse-embryonic fibroblasts (MEFs) with compound loss of both Ssbs. We further demonstrate that these MEFs show overexpression of unprocessed snRNAs which forces the cells to express alternative spliced variants of genes that are required for transcription and cell cycle regulation. This phenotype closely phenocopies loss of core integrator subunit, IntS11 as well as splicing components B and C. Interestingly, the phenotype gets partially restored by overexpression of wildtype Ssb1 but not by the Ssb-mutant defective in binding integrator subunit, IntS3, demonstrating the essential roles of Ssb1/2 in the maintenance of the Integrator complex and thereby, genomic stability.

ORGANISM(S): Mus musculus

PROVIDER: GSE155240 | GEO | 2022/03/11

REPOSITORIES: GEO

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Project description:The Integrator Complex (IC) is responsible for the 3'-end processing of the U1/U2 major spliceosome snRNAs. Maturation of these snRNAs is essential for the proper function of the major spliceosome. Also, there is accumulating evidence that the IC is necessary for normal vertebrate development, however no association with human disease has been reported yet. Three siblings presented with severe intellectual disability, cerebellar hypoplasia and periventricular nodular heterotopia (PNH) of unknown cause. Using whole genome sequencing (Complete Genomics), and filtering for recessive inheritance, we discovered co-segregating compound heterozygous mutations in Integrator Complex Subunit 8 (INTS8) in the three sibs. QRT-PCR showed 2-fold decreased INTS8 RNA levels and in patients’ fibroblasts lower levels of the catalytic subunits INTS4, INTS9 and INTS11 were observed on western blots, indicating that the integrity of the complex is disrupted. Northern blots showed significantly misprocessed levels of U1, U2 and U4. Pathway analysis (DAVID) of expression data (Affymetrix Gene Chip 1.0 ST exon arrays) of RNA extracted from patient fibroblasts showed enrichment of deregulated genes involved in mRNA splicing and posttranscriptional modification. Expression data of (developing) human brain structures (Allen Brain Atlas) show that expression of INTS8 peaks prenatally, especially in the ganglionic eminences, (sub)ventricular zone and hindbrain, similar to the developmental expression in mouse embryo (Emage databse). Interestingly, neuronal precursors from these areas migrate to the cortex and are compatible to affected regions in PNH and cerebellar hypoplasia in the patients. We propose that dysfunction of the Integrator Complex, through snRNA misprocessing and spliceosomal defects, leads to severely disrupted brain development in humans. We used RNA isolated from human cultured fibroblasts, cell-lines were obtained from 3 individuals with INTS8 mutations and from 3 unaffected individuals. R,Oegema Six Affymetrix Gene Chip 1.0 ST exon arrays (patients: 04RD83, 83RD247, 04RD84 ; controls: 81RD193, GMO2037C, 86RD677) were rma normalized on transcript level using the R package oligo (http://www.r-project.org). Top down regulated (n=402) or up regulated (n=281) genes with p values <0.02 were analyzed for enriched gene ontology terms (goterms_BP_all) using DAVID (medium stringency)(down: 329 DAVID IDs, up: 225 DAVID IDs).