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. 2011 Jan;31(2):328-41.
doi: 10.1128/MCB.00943-10. Epub 2010 Nov 15.

A subset of Drosophila integrator proteins is essential for efficient U7 snRNA and spliceosomal snRNA 3'-end formation

Nader Ezzeddine  1 , Jiandong Chen, Bernhard Waltenspiel, Brandon Burch, Todd Albrecht, Ming Zhuo, William D Warren, William F Marzluff, Eric J Wagner
Affiliations

A subset of Drosophila integrator proteins is essential for efficient U7 snRNA and spliceosomal snRNA 3'-end formation

Nader Ezzeddine et al. Mol Cell Biol. 2011 Jan.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
Design and assessment of a U7 snRNA misprocessing reporter. (A) Diagrammatic representation of the U7 snRNA gene, located within intron 4 of the Eip63E gene, which was cloned upstream of GFP. The endogenous U7 promoter, coding region, cleavage site, and 3′ box present in the minigene construct are depicted, as well as the predicted structures of both processed and misprocessed transcripts. αHDE, anti-HDE. (B) Drosophila S2 cells were treated with dsRNA (labeled in white), followed by transfection of the U7-GFP reporter. Bright-field images are in the top row, while corresponding GFP fluorescence is shown in the bottom row. (C) Western blot analysis of cell lysates treated with PTB, CPSF30 (negative controls) dsRNA, or dsRNA targeting Integrator 9 using anti-IntS9 (αIntS9) antibodies. The asterisk denotes a nonspecific cross-reacting band.
FIG. 2.
FIG. 2.
A non-snRNA promoter causes constitutive misprocessing of U7 snRNA and insensitivity to Integrator knockdown. (A) Bright-field and fluorescence images of Drosophila S2 cells treated with either control (Con.) dsRNA or dsRNA targeting Integrator 9 and then transfected with either an actin 5C-driven U7-GFP reporter or with the native U7-GFP reporter. (B) Lysates from the cells in panel A were analyzed by Western blotting using anti-Integrator 9, anti-RNAPII (loading control), or anti-GFP antibodies.
FIG. 3.
FIG. 3.
Characterization of the cis element requirements for U7 snRNA 3′-end processing. (A) Schematic of the scanning mutations within the 30 nucleotides downstream of the 3′ end of the mature U7 snRNA in the U7-GFP construct. Drosophila S2 cells were transfected with plasmids carrying mutations 1 to 6 (mt1 to mt6; top), and lysates were analyzed for snRNA misprocessing, as indicated by GFP expression, using anti-GFP antibodies or antibodies raised against RNAPII (con.). Both short and long exposures are shown for the GFP Western blot (bottom). (B) Schematic of the point mutations made within the 3′ terminal stem-loop of the U7 snRNA coding region (top) and Western blotting of cell extracts to quantitate GFP expression (bottom). The location of the mutated nucleotides (labeled mtA to mtG) are shaded. The lower blot represents an analysis similar to that described for panel A.
FIG. 4.
FIG. 4.
Knockdown of the Drosophila Integrator subunits causes misprocessing of the U7-GFP reporter. (A) Western blot analysis of lysates from Drosophila S2 cells treated with either control dsRNA targeting PTB or a specific dsRNA targeting production of Integrator 1, 11, or 12. RNAPII (center) or a cross-reacting band (asterisks) serves as a loading control. (B) Fluorescence images of cells treated with a specific dsRNA (labeled in white), then transfected with the U7-GFP reporter. (C) Western blot analysis of cell lysates from the cells shown in panel B. Lysates were probed with either anti-GFP antibodies or with anti-Symplekin (αSym.) antibodies as a control. (D) Fluorescence images of cells treated with dsRNA followed by transfection with the U7-GFP reporter. In this case the control dsRNA targeted LacZ and not PTB. (E) Western blot analysis of cell lysates from panel D.
FIG. 5.
FIG. 5.
Cleavage and polyadenylation factors and histone pre-mRNA processing factors do not affect U7 snRNA 3′ processing. (A) Alignment of sequences downstream of several Drosophila snRNA mature 3′ ends reveals a well-conserved polyadenylation signal sequence present downstream of nearly all of the Drosophila snRNA genes (shaded). (B) Fluorescence images of Drosophila S2 cells treated with dsRNA to knock down specific cleavage and polyadenylation factors (labeled in white), followed by transfection with either the U7-GFP minigene reporter or the histone H3-GFP minigene reporter.
FIG. 6.
FIG. 6.
Knockdown of Integrator subunits causes various degrees of misprocessing of endogenous spliceosomal snRNAs. (A) Histogram of real-time PCR experimental data generated using primer pairs designed to detect the presence of misprocessed (mis.) spliceosomal U1, U2, U4, or U5 snRNAs. Results are plotted as fold increases relative to control-treated cells and reflect expression normalized to an internal control gene (RpS17). All results are derived from biological triplicates, with error bars indicative of the standard deviations of the triplicate quantification. (B) Histogram of qRT-PCR quantitation of IntS3, IntS9, and IntS10 mRNA following dsRNA treatment. Levels represented are the averages of triplicate experiments normalized to an internal control (RpS17) and then normalized to control-treated cells. (C) Northern blot analysis for endogenous U4 snRNA and control (U6) snRNA from Drosophila S2 cells treated with either control dsRNA or dsRNA targeting IntS4. Both short and long exposures (ex.) of the U4 snRNA blot are shown. misproc., misprocessed. (D) Northern blot analysis of endogenous U4 snRNA expression in S2 cells treated with dsRNAs to knock down specific Integrator subunits.
FIG. 7.
FIG. 7.
Drosophila carrying germ line mutations in Integrator subunits accumulates misprocessed snRNA and fails to complete development. (A, top) Schematic of the location of a P element within the second exon of the Integrator 4 gene in strain P(lacW)l(1)G0095, as well as the locations of PCR primers in exon 1 (C) and exon 2 (D) and within the inverted terminal repeat of the P element (P). (Bottom) PCR amplification of genomic DNA isolated from either wild-type (wt) larvae or larvae homozygous for the P element within the Integrator 4 gene. (B) RT-PCR analysis of RNA isolated from the larvae described in panel A using primers specific for the IntS4 mRNA or RpS17 mRNA (control). +RT indicates cDNA reaction mixtures containing reverse transcriptase, and −RT indicates mock cDNA reaction mixtures lacking reverse transcriptase. (C) Northern blot analysis of endogenous U7 snRNA from either wild-type, U7EY11305, or IntS4 P element-containing third-instar larvae showing accumulation of misprocessed snRNA in the IntS4 mutant. (D) Results from qRT-PCR analysis of misprocessed U1 snRNA or U7 snRNA isolated from third-instar larvae from either homozygous IntS4 [P(lacW)l(1)G0095] or IntS7 (deflL) null mutants.
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