title: Functional analysis of the integrator subunit 12 identifies a microdomain that mediates activation of the Drosophila integrator complex.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Texas Medical School at Houston, Houston, Texas 77030, USA.
authors.forename: Jiandong
authors.initials: J
authors.lastname: Chen
authors.forename: Bernhard
authors.initials: B
authors.lastname: Waltenspiel
authors.forename: William D
authors.initials: WD
authors.lastname: Warren
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Wagner
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004330:Drosophila / Q000378:metabolism*; D029721:Drosophila Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D023281:Genomics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D008969:Molecular Sequence Data; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques
chemical_list: D029721:Drosophila Proteins; D018398:Homeodomain Proteins; C580721:IntS1 protein, Drosophila; C580720:IntS12 protein, Drosophila; D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; D049452:Green Fluorescent Proteins