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. 2013 Feb 15;288(7):4867-77.
doi: 10.1074/jbc.M112.425892. Epub 2013 Jan 3.

Functional analysis of the integrator subunit 12 identifies a microdomain that mediates activation of the Drosophila integrator complex

Jiandong Chen  1 , Bernhard Waltenspiel, William D Warren, Eric J Wagner
Affiliations

Functional analysis of the integrator subunit 12 identifies a microdomain that mediates activation of the Drosophila integrator complex

Jiandong Chen et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
Sensitive dual GFP reporters reveal snRNA misprocessing following IntS12 knockdown in Drosophila S2 cells. A, fluorescence and bright field (BF) images of dsRNA-treated S2 cells (upper panel) and schematic representations of the snRNA reporters (bottom panel). EGFP, enhanced GFP. B, Western blot analysis of cell lysates from A using anti-GFP or anti-IntS12 antibodies. A nonspecific band that cross-reacts with the IntS12 antibody was used as a loading control (Con.). C, graphical representation of quantitative real-time PCR of endogenous snRNA misprocessing. Shown are amplicons specific for non-processed snRNAs isolated from S2 cells treated with either IntS12#1 or IntS12#2 dsRNAs. Data are the average of triplicate independent experiments normalized to Rps17 mRNA and plotted as fold increase relative to LacZ-treated control cells.
FIGURE 2.
FIGURE 2.
RNAi-resistant IntS12* rescues dsRNA-induced U4 and U7 snRNA misprocessing. A, schematic of the features encoded in the Drosophila IntS12 (CG5491) gene showing the relative location of dsRNA#2 and the 188 silent site changes used to generate the IntS12* cDNA. B, Western blot analysis of lysates from cells transfected with myc-tagged IntS12 (wild-type) cDNA or IntS12* cDNA using anti-myc antibodies (left panel) or from cells transfected with myc vector or myc-tagged IntS12* followed by IntS12 dsRNA#2 treatment using anti-IntS12 antibodies (right panel). The asterisk represents a degradation product of the overexpressed IntS12*. Unt., untreated; Resc., rescue plasmid; VA, vector alone. C, Western blot analysis demonstrating dose-dependent rescue of the U7-GFP and U4-GFP misprocessing phenotype using the IntS12* cDNA following RNAi-mediated depletion. Lanes 2–7 are from S2 cells treated with IntS12 dsRNA#2 subsequently cotransfected with reporter plasmid and rescue plasmid DNAs at doses of rescue plasmid indicated in nanograms. D, representative fluorescence images of S2 cells treated as described in C. In all panels, an ∼60-kDa cross-reacting band with anti-IntS1 polyclonal antibodies is used as the loading control (Con.).
FIGURE 3.
FIGURE 3.
The N terminus of Drosophila IntS12 is required for snRNA 3′ end formation. A, schematic of IntS12* truncation and deletion constructs, which were designed on the basis of predicted domains. Relevant amino acid sequences are numbered. B, Western blot analysis of cell lysates isolated from S2 cells transiently transfected with plasmids encoding myc-tagged IntS12* proteins. C, representative fluorescence images of S2 cells treated with either control dsRNA or IntS12 dsRNA#2 followed by cotransfection of either the U4-GFP or U7-GFP reporters with the myc-tagged IntS12* cDNAs. D, Western blot analysis of cell lysates from C. Resc., rescue plasmid; VA, vector alone. E, schematic and Western blot analysis of three additional IntS12* deletion mutants cotransfected with the U4-GFP reporter. In all panels, an ∼60-kDa cross-reacting band with anti-IntS1 polyclonal antibodies is used as the loading control (Con.).
FIGURE 4.
FIGURE 4.
Mapping critical residues within the N-terminal IntS12 microdomain required for snRNA 3′ end formation. A, upper panel, alignment of the IntS12 N termini of several species. Residues highlighted in blue represent similar amino acids, and yellow highlights represent identical residues. The highlighted red box denotes the identified functional microdomain residues 16–45. The labeled amino acids are the subject of six different alanine-scanning mutants. B, Western blot analysis of cell lysates treated with either control dsRNA or IntS12 dsRNA#2 that were then cotransfected with U4-GFP reporter and IntS12* plasmids containing mutations as described in A using anti-GFP and anti-IntS12 antibodies. An ∼60-kDa cross-reacting band with anti-IntS1 polyclonal antibodies is used as the loading control (Con.). Resc., rescue plasmid; VA, vector alone.
FIGURE 5.
FIGURE 5.
Loss-of-function mutations in the IntS12 microdomain disrupt IntS12 interaction with endogenous integrator subunits. A, Western blot analysis of cell lysates from S2 cells treated with either control dsRNA or IntS12 dsRNA#2 followed by cotransfection of FLAG-tagged rescue plasmids and either the U4-GFP or U7-GFP reporter. Resc., rescue plasmid; VA, vector alone. B, quantitative real-time PCR measuring levels of misprocessed endogenous U2 or U5 snRNA in stable cell lines treated with dsRNA targeting IntS12. Control (Con.) represents S2 cells expressing FLAG only, and cell lines stably expressing FLAG-tagged IntS12 proteins are labeled on the x axis. All results are plotted as fold increase relative to LacZ dsRNA treatment and normalized to RpS17 mRNA levels. C, Western blot analysis of immunoprecipitations (IP) using anti-FLAG-agarose from nuclear extracts prepared from cell lines stably expressing FLAG-tagged IntS12* proteins. Inp, input. D, Western blot analysis of cell lysates from S2 cells treated with either control dsRNA or IntS12 dsRNA#2 followed by cotransfection with U4-GFP and FLAG-mCherry plasmids with or without the N-terminal 45 amino acids of IntS12. E, Western blot analysis of immunoprecipitations using anti-FLAG-agarose from nuclear extracts purified from cell lines stably expressing FLAG-tagged IntS12* proteins and FLAG-mCherry proteins with and without the IntS12 microdomain (N45). The upper panels show probes for endogenous IntS1/9, and the bottom panel shows probes with anti-FLAG antibody to confirm pull-down. In A and D, an ∼60-kDa cross-reacting band with anti-IntS1 polyclonal antibodies is used as the loading control.
FIGURE 6.
FIGURE 6.
The IntS12 microdomain is necessary and partially sufficient to mediate interaction with IntS1 in the absence of other integrator subunits. A, S. cerevisiae (AH109) was cotransformed with plasmids encoding hybrid proteins containing IntS12 fused to the Gal4 DNA activation domain (AD-IntS12) and each of the other integrator subunits fused to the Gal4 DNA binding domain (BD-IntSs). A dilution series of overnight cultures was spotted on either SC/-Leu/-Trp plates or the same media without histidine to test interaction. All BD constructs were tested for autoactivation. B, similar A, except that AH109 yeast was transformed with full-length IntS12 containing mutations 3 or 5, a deletion of the N-terminal 45 amino acids (ΔN), or the first 45 amino acids of IntS12 (N45). C, Western blot analysis confirming the expression of HA-tagged IntS12 constructs in yeast strain AH109. Con., control.
FIGURE 7.
FIGURE 7.
Expression of Drosophila IntS1 and IntS12 is interdependent, and stability of IntS1 requires an intact IntS12 microdomain. A, Western blot analysis of endogenous integrator subunit expression from S2 cells treated with various dsRNA targeting IntSs. An ∼60-kDa cross-reacting band with anti-IntS1 polyclonal antibodies is used as the loading control (Con.). B, Western blot analysis of endogenous IntS1 expression in S2 cells stably expressing FLAG-tagged IntS12* proteins containing mutations within the IntS12 microdomain. An ∼130-kDa cross-reacting band with anti-IntS1 polyclonal antibodies is used as the loading control. Resc., rescue plasmid; VA, vector alone.
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