RESUMEN
The mechanisms of pre-mRNA splicing regulation are poorly understood. Here we dissect how the Saccharomyces cerevisiae ribosomal L30 protein blocks splicing of its pre-mRNA upon binding a kink-turn structure including the 5' splice site. We show that L30 binds the nascent RPL30 transcript without preventing recognition of the 5' splice site by U1 snRNP but blocking U2 snRNP association with the branch site. Interaction of the factors BBP and Mud2 with the intron, relevant for U2 snRNP recruitment, is not affected by L30. Furthermore, the functions of neither the DEAD-box protein Sub2 in the incipient spliceosome nor the U2 snRNP factor Cus2 on branch site recognition are required for L30 inhibition. These findings contrast with the effects caused by binding a heterologous protein to the same region, completely blocking intron recognition. Collectively, our data suggest that L30 represses a spliceosomal rearrangement required for U2 snRNP association with the transcript.
Asunto(s)
Galectina 3/farmacología , Empalme del ARN , Ribonucleoproteína Nuclear Pequeña U2/química , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Intrones , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , ARN de Hongos/química , ARN de Hongos/efectos de los fármacos , ARN de Hongos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción GenéticaRESUMEN
Pre-mRNA splicing is catalyzed by the spliceosome, and its control is essential for correct gene expression. While splicing repressors typically interfere with transcript recognition by spliceosomal components, the yeast protein L30 blocks spliceosomal rearrangements required for the engagement of U2 snRNP (small ribonucleoprotein particle) to its own transcript RPL30. Using a mutation in the RPL30 binding site that disrupts this repression, we have taken a genetic approach to reveal that regulation of splicing is restored in this mutant by deletion of the cap-binding complex (CBC) component Cbp80. Indeed, our data indicate that Cbp80 plays distinct roles in the recognition of the intron by U1 and U2 snRNP. It promotes the initial 5' splice site recognition by U1 and, independently, facilitates U2 recruitment, depending on sequences located in the vicinity of the 5' splice site. These results reveal a novel function for CBC in splicing and imply that these molecular events can be the target of a splicing regulator.
Asunto(s)
Proteínas Nucleares/metabolismo , Proteínas de Unión a Caperuzas de ARN/metabolismo , Empalme del ARN , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Proteínas Ribosómicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN/genética , Exones , Eliminación de Gen , Genes Fúngicos , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/genética , Conformación de Ácido Nucleico , Proteínas de Unión a Caperuzas de ARN/genética , Precursores del ARN/química , Precursores del ARN/genética , Precursores del ARN/metabolismo , Sitios de Empalme de ARN , ARN de Hongos/química , ARN de Hongos/genética , ARN de Hongos/metabolismo , Proteínas Ribosómicas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Empalmosomas/metabolismoRESUMEN
In cirrhotic livers, increased resistance to portal flow, in part due to an exaggerated response to vasoconstrictors, is the primary factor in the pathophysiology of portal hypertension. Our aim was to evaluate the response of the intrahepatic circulation of cirrhotic rat livers to the alpha(1)-adrenergic vasoconstrictor methoxamine and the mechanisms involved in its regulation. A portal perfusion pressure dose-response curve to methoxamine was performed in control and cirrhotic rat livers preincubated with vehicle, the nitric oxide synthase blocker N(G)-nitro-L-arginine (L-NNA), indomethacin cyclooxygenase (COX) inhibitor, L-NNA + indomethacin, or the thromboxane (TX) A(2) receptor blocker SQ 29,548. TXA(2) production, COX-1 and COX-2 mRNA expression, and immunostaining for TXA(2) synthase were evaluated. Cirrhotic livers exhibited a hyperresponse to methoxamine associated with overexpression of COX-2 and TXA(2) synthase as well as with increased production of TXA(2). The hyperresponse to methoxamine of cirrhotic livers disappeared by COX inhibition with indomethacin but not after NO inhibition. SQ 29,548 also corrected the hyperresponse of cirrhotic livers to methoxamine. In conclusion, COX-derived prostanoids, mainly TXA(2), play a major role in regulating the response of cirrhotic livers to methoxamine.