RESUMO
Tumour cell plasticity is a major barrier to the efficacy of targeted cancer therapies but the mechanisms that mediate it are poorly understood. Here, we identify dysregulated RNA splicing as a key driver of tumour cell dedifferentiation in colorectal cancer (CRC). We find that Apc-deficient CRC cells have dysregulated RNA splicing machinery and exhibit global rewiring of RNA splicing. We show that the splicing factor SRSF1 controls the plasticity of tumour cells by controlling Kras splicing and is required for CRC invasion in a mouse model of carcinogenesis. SRSF1 expression maintains stemness in human CRC organoids and correlates with cancer stem cell marker expression in human tumours. Crucially, partial genetic downregulation of Srsf1 does not detrimentally affect normal tissue homeostasis, demonstrating that tumour cell plasticity can be differentially targeted. Thus, our findings link dysregulation of the RNA splicing machinery and control of tumour cell plasticity.
Assuntos
Plasticidade Celular , Neoplasias Colorretais , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Plasticidade Celular/genética , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Camundongos , Splicing de RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismoRESUMO
Y RNAs (84-112 nt) are non-coding RNAs transcribed by RNA polymerase III and are characterized by a distinctive secondary structure. Human Y RNAs interact with the autoimmune proteins SSB and RO60 that together form a ribonucleoprotein (RNP) complex termed RoRNP and Y RNAs also perform regulatory roles in DNA and RNA replication and stability, which has major implications for diseases including cancer. During cellular stress and apoptosis, Y RNAs are cleaved into 3' and 5' end fragments termed Y RNA-derived small RNAs (ysRNAs). Although some ysRNA functions in stress, apoptosis and cancer have been reported, their fundamental biogenesis has not been described. Here we report that 3' end RNY5 cleavage is structure dependent. In high throughput mutagenesis experiments, cleavage occurred between the 2nd and 3rd nt above a double stranded stem comprising high GC content. We demonstrate that an internal loop above stem S3 is critical for producing 3' end ysRNAs (31 nt) with mutants resulting in longer or no ysRNAs. We show a UGGGU sequence motif at position 22 of RNY5 is critical for producing 5' end ysRNAs (22-25 nt). We show that intact RO60 is critical for ysRNA biogenesis. We conclude that ribonuclease L (RNASEL) contributes to Y RNA cleavage in mouse embryonic fibroblasts but is not the only endoribonuclease important in human cells.
Assuntos
RNA não Traduzido , Ribonucleoproteínas , Animais , Fibroblastos/metabolismo , Camundongos , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA , RNA não Traduzido/genética , Ribonucleoproteínas/metabolismoRESUMO
Current therapeutic options for treating colorectal cancer have little clinical efficacy and acquired resistance during treatment is common, even following patient stratification. Understanding the mechanisms that promote therapy resistance may lead to the development of novel therapeutic options that complement existing treatments and improve patient outcome. Here, we identify RAC1B as an important mediator of colorectal tumourigenesis and a potential target for enhancing the efficacy of EGFR inhibitor treatment. We find that high RAC1B expression in human colorectal cancer is associated with aggressive disease and poor prognosis and deletion of Rac1b in a mouse colorectal cancer model reduces tumourigenesis. We demonstrate that RAC1B interacts with, and is required for efficient activation of the EGFR signalling pathway. Moreover, RAC1B inhibition sensitises cetuximab resistant human tumour organoids to the effects of EGFR inhibition, outlining a potential therapeutic target for improving the clinical efficacy of EGFR inhibitors in colorectal cancer.
Assuntos
Neoplasias Colorretais/etiologia , Neoplasias Colorretais/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Antineoplásicos Imunológicos/farmacologia , Carcinogênese , Linhagem Celular Tumoral , Cetuximab/farmacologia , Neoplasias Colorretais/genética , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuropeptídeos/deficiência , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Transdução de Sinais , Regulação para Cima , Via de Sinalização Wnt , Proteínas rac1 de Ligação ao GTP/deficiência , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
The different RNA populations within the cell, or the 'RNAome', form a complex and integral layer of gene regulation. In particular, small non-coding RNAs such as microRNAs have been shown to regulate as much as half of all genes expressed in mammals. The recent discovery of novel small RNAs derived from tRNAs, snoRNAs and Y RNAs, as well as the development of techniques that can reduce sequencing bias of these molecules, is slowly helping us to unveil the secrets of the genome.
Assuntos
Genoma , RNA/genética , Animais , MamíferosRESUMO
Non-coding RNAs have emerged as key regulators in diverse cellular processes. Y RNAs are â¼100-nucleotide-long non-coding RNAs that show high conservation in metazoans. Human Y RNAs are known to bind to the Ro60 and La proteins to form the Ro ribonucleoprotein complex. Their main biological function appears to be in mediating the initiation of chromosomal DNA replication, regulating the autoimmune protein Ro60, and generating smaller RNA fragments following cellular stress, although the precise molecular mechanisms underlying these functions remain elusive. Here, we aim to review the most recent literature on Y RNAs and gain insight into the function of these intriguing molecules.
Assuntos
Replicação do DNA , DNA/metabolismo , RNA não Traduzido/metabolismo , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Citoplasmático Pequeno/genética , RNA Citoplasmático Pequeno/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMO
BACKGROUND: The use of nucleic acid-modifying enzymes has driven the rapid advancement in molecular biology. Understanding their function is important for modifying or improving their activity. However, functional analysis usually relies upon low-throughput experiments. Here we present a method for functional analysis of nucleic acid-modifying enzymes using next generation sequencing. FINDINGS: We demonstrate that sequencing data of libraries generated by RNA ligases can reveal novel secondary structure preferences of these enzymes, which are used in small RNA cloning and library preparation for NGS. Using this knowledge we demonstrate that the cloning bias in small RNA libraries is RNA ligase-dependent. We developed a high definition (HD) protocol that reduces the RNA ligase-dependent cloning bias. The HD protocol doubled read coverage, is quantitative and found previously unidentified microRNAs. In addition, we show that microRNAs in miRBase are those preferred by the adapters of the main sequencing platform. CONCLUSIONS: Sequencing bias of small RNAs partially influenced which microRNAs have been studied in depth; therefore most previous small RNA profiling experiments should be re-evaluated. New microRNAs are likely to be found, which were selected against by existing adapters. Preference of currently used adapters towards known microRNAs suggests that the annotation of all existing small RNAs, including miRNAs, siRNAs and piRNAs, has been biased.
RESUMO
Y RNAs are approximately 100 nucleotide long conserved cytoplasmic non-coding RNAs, which produce smaller RNA fragments during apoptosis. Here we show that these smaller RNA molecules are also produced in non-stressed cells and in a range of human cancerous and non-cancerous cell types. Recent reports have speculated that the cleavage products of Y RNAs enter the microRNA pathway. We tested this hypothesis and found that Y5 and Y3 RNA fragments are Dicer independent, they are in different complexes than microRNAs and that they are not co-immunoprecipitated with Ago2. Therefore we conclude that Y RNA fragments do not enter the microRNA pathway.