RESUMEN
The Drosophila integrator complex consists of 14 subunits that associate with the C terminus of Rpb1 and catalyze the endonucleolytic cleavage of nascent snRNAs near their 3' ends. Although disruption of almost any integrator subunit causes snRNA misprocessing, very little is known about the role of the individual subunits or the network of structural and functional interactions that exist within the complex. Here we developed an RNAi rescue assay in Drosophila S2 cells to identify functional domains within integrator subunit 12 (IntS12) required for snRNA 3' end formation. Surprisingly, the defining feature of the Ints12 protein, a highly conserved and centrally located plant homeodomain finger domain, is not required for reporter snRNA 3' end cleavage. Rather, we find a small, 45-amino acid N-terminal microdomain to be both necessary and nearly sufficient for snRNA biogenesis in cells depleted of endogenous IntS12 protein. This IntS12 microdomain can function autonomously, restoring full integrator processing activity when introduced into a heterologous protein. Moreover, mutations within the microdomain not only disrupt IntS12 function but also abolish binding to other integrator subunits. Finally, the IntS12 microdomain is sufficient to interact and stabilize the putative scaffold integrator subunit, IntS1. Collectively, these results identify an unexpected interaction between the largest and smallest integrator subunits that is essential for the 3' end formation of Drosophila snRNA.
Asunto(s)
Proteínas de Drosophila/química , Drosophila/metabolismo , Proteínas de Homeodominio/química , Secuencia de Aminoácidos , Animales , Línea Celular , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiología , Genómica , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/fisiología , Datos de Secuencia Molecular , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , ARN Nuclear Pequeño/metabolismo , Homología de Secuencia de Aminoácido , Transcripción Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Formation of the 3' end of RNA polymerase II-transcribed snRNAs requires a poorly understood group of proteins called the Integrator complex. Here we used a fluorescence-based read-through reporter that expresses GFP in response to snRNA misprocessing and performed a genome-wide RNAi screen in Drosophila S2 cells to identify novel factors required for snRNA 3'-end formation. In addition to the known Integrator complex members, we identified Asunder and CG4785 as additional Integrator subunits. Functional and biochemical experiments revealed that Asunder and CG4785 are additional core members of the Integrator complex. We also identified a conserved requirement in both fly and human snRNA 3'-end processing for cyclin C and Cdk8 that is distinct from their function in the Mediator Cdk8 module. Moreover, we observed biochemical association between Integrator proteins and cyclin C/Cdk8, and that overexpression of a kinase-dead Cdk8 causes snRNA misprocessing. These data functionally define the Drosophila Integrator complex and demonstrate an additional function for cyclin C/Cdk8 unrelated to its function in Mediator.
Asunto(s)
Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ciclina C/metabolismo , Quinasa 8 Dependiente de Ciclina/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Animales , Línea Celular , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Complejo Mediador/genética , Complejo Mediador/metabolismo , Procesamiento de Término de ARN 3' , Interferencia de ARN , Especificidad de la EspecieRESUMEN
Proper gene expression relies on a class of ubiquitously expressed, uridine-rich small nuclear RNAs (snRNAs) transcribed by RNA polymerase II (RNAPII). Vertebrate snRNAs are transcribed from a unique promoter, which is required for proper 3'-end formation, and cleavage of the nascent transcript involves the activity of a poorly understood set of proteins called the Integrator complex. To examine 3'-end formation in Drosophila melanogaster, we developed a cell-based reporter that monitors aberrant 3'-end formation of snRNA through the gain in expression of green fluorescent protein (GFP). We used this reporter in Drosophila S2 cells to determine requirements for U7 snRNA 3'-end formation and found that processing was strongly dependent upon nucleotides located within the 3' stem-loop as well as sequences likely to comprise the Drosophila equivalent of the vertebrate 3' box. Substitution of the actin promoter for the snRNA promoter abolished proper 3'-end formation, demonstrating the conserved requirement for an snRNA promoter in Drosophila. We tested the requirement for all Drosophila Integrator subunits and found that Integrators 1, 4, 9, and 11 were essential for 3'-end formation and that Integrators 3 and 10 may be dispensable for processing. Depletion of cleavage and polyadenylation factors or of histone pre-mRNA processing factors did not affect U7 snRNA processing efficiency, demonstrating that the Integrator complex does not share components with the mRNA 3'-end processing machinery. Finally, flies harboring mutations in either Integrator 4 or 7 fail to complete development and accumulate significant levels of misprocessed snRNA in the larval stages.
Asunto(s)
Regiones no Traducidas 3' , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , ARN Nuclear Pequeño/genética , Empalmosomas/metabolismo , Animales , Secuencia de Bases , Células Cultivadas , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Humanos , Datos de Secuencia Molecular , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Interferencia de ARN , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Nuclear Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Empalmosomas/genéticaRESUMEN
This review describes the present state of a project to identify and characterize novel nervous system proteins by using monoclonal antibodies (mAbs) against the Drosophila brain. Some 1,000 hybridoma clones were generated by injection of homogenized Drosophila brains or heads into mice and fusion of their spleen cells with myeloma cells. Testing the mAbs secreted by these clones identified a library of about 200 mAbs, which selectively stain specific structures of the Drosophila brain. Using the approach "from antibody to gene", several genes coding for novel proteins of the presynaptic terminal were cloned and characterized. These include the "cysteine string protein" gene (Csp, mAb ab49), the "synapse-associated protein of 47 kDa" gene (Sap47, mAbs nc46 and nb200), and the "Bruchpilot" gene (brp, mAb nc82). By a "candidate" approach, mAb nb33 was shown to recognize the pigment dispersing factor precursor protein. mAbs 3C11 and pok13 were raised against bacterially expressed Drosophila synapsin and calbindin-32, respectively, after the corresponding cDNAs had been isolated from an expression library by using antisera against mammalian proteins. Recently, it was shown that mAb aa2 binds the Drosophila homolog of "epidermal growth factor receptor pathway substrate clone 15" (Eps15). Identification of the targets of mAbs na21, ab52, and nb181 is presently attempted. Here, we review the available information on the function of these proteins and present staining patterns in the Drosophila brain for classes of mAbs that either bind differentially in the eye, in neuropil, in the cell-body layer, or in small subsets of neurons. The prospects of identifying the corresponding antigens by various approaches, including protein purification and mass spectrometry, are discussed.