RESUMEN
La-Related Protein 1 (LARP1) is an RNA-binding protein that regulates the stability and translation of mRNAs encoding the translation machinery, including ribosomal proteins and translation factors. These mRNAs are characterized by a 5'-terminal oligopyrimidine (TOP) motif that coordinates their temporal and stoichiometric expression. While LARP1 represses TOP mRNA translation via the C-terminal DM15 region, the role of the N-terminal La-Module in the recognition and translational regulation of TOP mRNAs remains elusive. Herein we show that the LARP1 La-Module also binds TOP motifs, although in a cap-independent manner. We also demonstrate that it recognizes poly(A) RNA. Further, our data reveal that the LARP1 La-Module can simultaneously engage TOP motifs and poly(A) RNA. These results evoke an intriguing molecular mechanism whereby LARP1 could regulate translation and stabilization of TOP transcripts.
Asunto(s)
Autoantígenos/química , Autoantígenos/metabolismo , Sitios de Unión , Dominios y Motivos de Interacción de Proteínas , ARN Mensajero/química , ARN Mensajero/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Regiones no Traducidas 5' , Autoantígenos/genética , Regulación de la Expresión Génica , Humanos , Modelos Biológicos , Motivos de Nucleótidos , Poli A , Unión Proteica , Procesamiento Proteico-Postraduccional , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , Ribonucleoproteínas/genética , Antígeno SS-BRESUMEN
The 5'terminal oligopyrimidine (5'TOP) motif is a cis-regulatory RNA element located immediately downstream of the 7-methylguanosine [m7G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5'TOP motif, a cap analog (m7GTP), and a capped cytidine (m7GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5'TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5'TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis.
Asunto(s)
Autoantígenos/química , Autoantígenos/metabolismo , Factor 4E Eucariótico de Iniciación/antagonistas & inhibidores , Factor 4F Eucariótico de Iniciación/antagonistas & inhibidores , Secuencia de Oligopirimidina en la Región 5' Terminal del ARN , ARN Mensajero/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Inmunoprecipitación de Cromatina , Cristalografía por Rayos X , Regulación de la Expresión Génica , Modelos Moleculares , Unión Proteica , Biosíntesis de Proteínas , Conformación Proteica , Estabilidad del ARN , Antígeno SS-BRESUMEN
Human polyomaviruses are generally latent but can be reactivated in patients whose immune systems are suppressed. Unfortunately, current therapeutics for diseases associated with polyomaviruses are non-specific, have undefined mechanisms of action, or exacerbate the disease. We previously reported on a class of dihydropyrimidinones that specifically target a polyomavirus-encoded protein, T antigen, and/or inhibit a cellular chaperone, Hsp70, that is required for virus replication. To improve the antiviral activity of the existing class of compounds, we performed Biginelli and modified multi-component reactions to obtain new 3,4-dihydropyrimidin-2(1H)-ones and -thiones for biological evaluation. We also compared how substituents at the N-1 versus N-3 position in the pyrimidine affect activity. We discovered that AMT580-043, a N-3 alkylated dihydropyrimidin-2(1H)-thione, inhibits the replication of a disease-causing polyomavirus in cell culture more potently than an existing drug, cidofovir.
Asunto(s)
Antivirales/farmacología , Poliomavirus/efectos de los fármacos , Pirimidinonas/farmacología , Animales , Antivirales/química , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Humanos , Pirimidinonas/química , Relación Estructura-ActividadRESUMEN
RNA-binding proteins (RBPs) are increasingly identified as post-transcriptional drivers of cancer progression. The RBP LARP1 is an mRNA stability regulator, and elevated expression of the protein in hepatocellular and lung cancers is correlated with adverse prognosis. LARP1 associates with an mRNA interactome that is enriched for oncogenic transcripts. Here we explore the role of LARP1 in epithelial ovarian cancer, a disease characterized by the rapid acquisition of resistance to chemotherapy through the induction of pro-survival signalling. We show, using ovarian cell lines and xenografts, that LARP1 is required for cancer cell survival and chemotherapy resistance. LARP1 promotes tumour formation in vivo and maintains cancer stem cell-like populations. Using transcriptomic analysis following LARP1 knockdown, cross-referenced against the LARP1 interactome, we identify BCL2 and BIK as LARP1 mRNA targets. We demonstrate that, through an interaction with the 3' untranslated regions (3' UTRs) of BCL2 and BIK, LARP1 stabilizes BCL2 but destabilizes BIK with the net effect of resisting apoptosis. Together, our data indicate that by differentially regulating the stability of a selection of mRNAs, LARP1 promotes ovarian cancer progression and chemotherapy resistance.