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HnRNP L regulates the tumorigenic capacity of lung cancer xenografts in mice via caspase-9 pre-mRNA processing.

ABSTRACT: Caspase-9 is involved in the intrinsic apoptotic pathway and suggested to play a role as a tumor suppressor. Little is known about the mechanisms governing caspase-9 expression, but post-transcriptional pre-mRNA processing generates 2 splice variants from the caspase-9 gene, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b. Here we demonstrate that the ratio of caspase-9 splice variants is dysregulated in non-small cell lung cancer (NSCLC) tumors. Mechanistic analysis revealed that an exonic splicing silencer (ESS) regulated caspase-9 pre-mRNA processing in NSCLC cells. Heterogeneous nuclear ribonucleoprotein L (hnRNP L) interacted with this ESS, and downregulation of hnRNP L expression induced an increase in the caspase-9a/9b ratio. Although expression of hnRNP L lowered the caspase-9a/9b ratio in NSCLC cells, expression of hnRNP L produced the opposite effect in non-transformed cells, suggesting a post-translational modification specific for NSCLC cells. Indeed, Ser52 was identified as a critical modification regulating the caspase-9a/9b ratio. Importantly, in a mouse xenograft model, downregulation of hnRNP L in NSCLC cells induced a complete loss of tumorigenic capacity that was due to the changes in caspase-9 pre-mRNA processing. This study therefore identifies a cancer-specific mechanism of hnRNP L phosphorylation and subsequent lowering of the caspase-9a/9b ratio, which is required for the tumorigenic capacity of NSCLC cells.

SUBMITTER: Goehe RW

PROVIDER: S-EPMC2964989 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Modulation of pre-mRNA structure by hnRNP proteins regulates alternative splicing of <i>MALT1</i>.

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Nuclear pre-mRNA 3'-end processing regulates synapse and axon development in C. elegans.

Project description:Nuclear pre-mRNA 3'-end processing is vital for the production of mature mRNA and the generation of the 3' untranslated region (UTR). However, the roles and regulation of this event in cellular development remain poorly understood. Here, we report the function of a nuclear pre-mRNA 3'-end processing pathway in synapse and axon formation in C. elegans. In a genetic enhancer screen for synaptogenesis mutants, we identified a novel polyproline-rich protein, Synaptic defective enhancer-1 (SYDN-1). Loss of function of sydn-1 causes abnormal synapse and axon development, and displays striking synergistic interactions with several genes that regulate specific aspects of synapses. SYDN-1 is required in neurons and localizes to distinct regions of the nucleus. Through a genetic suppressor screen, we found that the neuronal defects of sydn-1 mutants are suppressed by loss of function in Polyadenylation factor subunit-2 (PFS-2), a conserved WD40-repeat protein that interacts with multiple subcomplexes of the pre-mRNA 3'-end processing machinery. PFS-2 partially colocalizes with SYDN-1, and SYDN-1 influences the nuclear abundance of PFS-2. Inactivation of several members of the nuclear 3'-end processing complex suppresses sydn-1 mutants. Furthermore, lack of sydn-1 can increase the activity of 3'-end processing. Our studies provide in vivo evidence for pre-mRNA 3'-end processing in synapse and axon development and identify SYDN-1 as a negative regulator of this cellular event in neurons.

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Project description:The human hnRNP C is a ubiquitous cellular protein involved in mRNA maturation. Recently, we have shown that this protein specifically recognizes uridine (U) pentamers through its single RNA recognition motif (RRM). However, a large fraction of natural RNA targets of hnRNP C consists of much longer contiguous uridine stretches. To understand how these extended sites are recognized, we studied the binding of the RRM to U-tracts of 8-11 bases. In vivo investigation of internal translation activation of unr (upstream of N-ras) mRNA indicates that the conservation of the entire hnRNP C binding site, UC(U)8, is required for hnRNP C-dependent IRES activation. The assays further suggest a synergistic interplay between hnRNP C monomers, dependent on the protein's ability to oligomerize. In vitro spectroscopic and thermodynamic analyses show that isolated RRMs bind to (U)11 oligomers as dimers. Structural modeling of a ternary double-RRM/RNA complex indicates additionally that two RRM copies can be accommodated on the canonical sequence UC(U)8. The proposed tandem RRM binding is in very good agreement with the transcriptome-wide recognition of extended U-tracts by full-length hnRNP C, which displays a cross-linking pattern consistent with a positively cooperative RRM dimer binding model.

| S-EPMC4604433 | biostudies-literature

Alternative pre-mRNA processing regulates cell-type specific expression of the IL4l1 and NUP62 genes.

Project description:BACKGROUND:Given the complexity of higher organisms, the number of genes encoded by their genomes is surprisingly small. Tissue specific regulation of expression and splicing are major factors enhancing the number of the encoded products. Commonly these mechanisms are intragenic and affect only one gene. RESULTS:Here we provide evidence that the IL4I1 gene is specifically transcribed from the apparent promoter of the upstream NUP62 gene, and that the first two exons of NUP62 are also contained in the novel IL4I1_2 variant. While expression of IL4I1 driven from its previously described promoter is found mostly in B cells, the expression driven by the NUP62 promoter is restricted to cells in testis (Sertoli cells) and in the brain (e.g., Purkinje cells). Since NUP62 is itself ubiquitously expressed, the IL4I1_2 variant likely derives from cell type specific alternative pre-mRNA processing. CONCLUSION:Comparative genomics suggest that the promoter upstream of the NUP62 gene originally belonged to the IL4I1 gene and was later acquired by NUP62 via insertion of a retroposon. Since both genes are apparently essential, the promoter had to serve two genes afterwards. Expression of the IL4I1 gene from the "NUP62" promoter and the tissue specific involvement of the pre-mRNA processing machinery to regulate expression of two unrelated proteins indicate a novel mechanism of gene regulation.

| S-EPMC1198218 | biostudies-literature

An intron element modulating 5' splice site selection in the hnRNP A1 pre-mRNA interacts with hnRNP A1.

Project description:The hnRNP A1 pre-mRNA is alternatively spliced to yield the A1 and A1b mRNAs, which encode proteins differing in their ability to modulate 5' splice site selection. Sequencing a genomic portion of the murine A1 gene revealed that the intron separating exon 7 and the alternative exon 7B is highly conserved between mouse and human. In vitro splicing assays indicate that a conserved element (CE1) from the central portion of the intron shifts selection toward the distal donor site when positioned in between the 5' splice sites of exon 7 and 7B. In vivo, the CE1 element promotes exon 7B skipping. A 17-nucleotide sequence within CE1 (CE1a) is sufficient to activate the distal 5' splice site. RNase T1 protection/immunoprecipitation assays indicate that hnRNP A1 binds to CE1a, which contains the sequence UAGAGU, a close match to the reported optimal A1 binding site, UAGGGU. Replacing CE1a by different oligonucleotides carrying the sequence UAGAGU or UAGGGU maintains the preference for the distal 5' splice site. In contrast, mutations in the AUGAGU sequence activate the proximal 5' splice site. In support of a direct role of the A1-CE1 interaction in 5'-splice-site selection, we observed that the amplitude of the shift correlates with the efficiency of A1 binding. Whereas addition of SR proteins abrogates the effect of CE1, the presence of CE1 does not modify U1 snRNP binding to competing 5' splice sites, as judged by oligonucleotide-targeted RNase H protection assays. Our results suggest that hnRNP A1 modulates splice site selection on its own pre-mRNA without changing the binding of U1 snRNP to competing 5' splice sites.

| S-EPMC232024 | biostudies-other

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