Impaired spliceosomal UsnRNP assembly leads to Sm mRNA down-regulation and Sm protein degradation

Abstract

Specialized assembly factors facilitate the formation of many macromolecular complexes in vivo. The formation of Sm core structures of spliceosomal U-rich small nuclear ribonucleoprotein particles (UsnRNPs) requires assembly factors united in protein arginine methyltransferase 5 (PRMT5) and survival motor neuron (SMN) complexes. We demonstrate that perturbations of this assembly machinery trigger complex cellular responses that prevent aggregation of unassembled Sm proteins. Inactivation of the SMN complex results in the initial tailback of Sm proteins on the PRMT5 complex, followed by down-regulation of their encoding mRNAs. In contrast, reduction of pICln, a PRMT5 complex subunit, leads to the retention of newly synthesized Sm proteins on ribosomes and their subsequent lysosomal degradation. Overexpression of Sm proteins under these conditions results in a surplus of Sm proteins over pICln, promoting their aggregation. Our studies identify an elaborate safeguarding system that prevents individual Sm proteins from aggregating, contributing to cellular UsnRNP homeostasis.

Figure 1.

SMN knockdown results in mislocalization of SMN complex and UsnRNPs. (A) Lentivirus-mediated knockdown of SMN (shSMN). Western blot analysis of doxycycline-inducible shRNA knockdown of SMN, using tubulin as the normalization control. Quantification of the blot is shown in the bottom panel, with black and gray bars indicating control and SMN knockdown, respectively. (B) FACS analysis of control uninduced cells (top), shSMN at 120 h (middle) and shSMN at 144 h (bottom) after doxycycline induction of shRNA, after staining with annexin V (FL4-A channel) and propidium iodide (FL3-A channel). Left, gating of all cells (brown); right, distribution of necrotic (bottom right quadrant), early apoptotic (top right quadrant), and apoptotic (top left quadrant) cells along with the respective quantification. (C) Western blot using antibodies specific to SMN to validate knockdown in cells used for FACS analysis, with tubulin as loading control. (D) Confocal images of control cells (top) and shSMN cells (bottom) stained using antibodies against SMN, Gemin5, SmD1/B/B'/D3 (Y12 antibody), and coilin as well as DAPI for nuclear staining. White arrowheads in SMN and Gemin5 images indicate predominant staining pattern observed in cytoplasm (shSMN cells) and Cajal bodies (CBs) and cytoplasm (control cells). White arrowheads in Sm images indicate CBs, splicing speckles (control), and speckles (shSMN). White arrowheads in coilin images indicate intact CBs (control) and disintegrated CBs/mislocalized coilin (shSMN).

Figure 2.

Knockdown of SMN causes Sm protein tailback on pICln. (A) Bar graphs representing normalized log₂ fold change in expression (control/shSMN) of UsnRNP-related proteins as well as those of SMN complex and pICln (circles in scatter plot; B) in pSILAC experiments. Dark- and light-gray bars represent two independent replicates. (B) Normalized values of fold change in protein expression during SMN depletion, from two independent pSILAC experiments. Circles indicate proteins involved in UsnRNP pathway that are listed in the bar graph in A. Arrow in the top right quadrant indicates SMN; arrow in the center indicates Sm proteins (gray area). Red, blue, and gray colors indicate high, moderate, or insignificant changes in expression, respectively. (Right) Western blot showing steady-state levels of Sm proteins (sDMA SmB/B', D1, D3) in control and shSMN samples. (C) [³⁵S]methionine metabolic labeling and immunoprecipitation (IP) using antibodies against symmetrically dimethylated Sm proteins, pICln, Gemin5, and m₃G/m⁷G-cap. (Top) Autoradiography of immunoprecipitated samples from control and SMN knockdown cells. (Bottom) Corresponding Western blot controls for immunoprecipitation with molecular weight (in kD) from the marker indicated on the left of the image. (D) Quantification of the autoradiography image shown in C, from three independent biological experiments for Sm, Gemin5, and m₃G/m⁷G-cap IPs and five independent biological experiments for pICln IP. Black and gray bars represent control and SMN knockdown conditions, respectively. Error bars represent standard error (SE); *, P < 0.05 calculated using Student’s t test.

Figure 3.

Destabilization of Sm transcripts after extended inhibition of the late assembly phase. (A) Normalized fold change of transcripts encoding the indicated Sm proteins, SMN, and Gemin5, as well as U1 and U2 snRNAs in control cells (black bar) and SMN knockdown cells (gray bars), respectively, 144 h after doxycycline induction. (Top) Western blot analysis showing knockdown of SMN, with tubulin as loading control. (B) Normalized fold change of random transcripts (PI15, TOP3b, CPOX, hTR, and ASNS) that are unrelated to UsnRNP biogenesis as well as LSm encoding transcripts (LSm 2, 4,10, 11), PHAX and SNUP1. (C) qRT-PCR analysis of recovery of Sm encoding transcripts in control (black bars) and SMN knockdown (gray bars) cells upon treatment with 5-FU, an exosome inhibitor. Expression normalized to control cells treated with 5-FU. (Top) Western blot analysis showing knockdown of SMN, with tubulin as loading control. (D) Normalized fold change of transcripts encoding the indicated Sm proteins, SMN, and Gemin5, as well as U1 and U2 snRNAs in control cells (black bar) and SMN knockdown cells (gray bars) 168 h after doxycycline induction. (Top) Western blot analysis showing knockdown of SMN, with tubulin as loading control. Error bars indicate SE (n = 3, three independent biological experiments). **, P < 0.0005; *, P < 0.05, Student’s t test. GAPDH mRNA was used as a control for normalization.

Figure 4.

Sm proteins are down-regulated in the absence of the pICln. (A) FACS analysis of control (top) and pICln knockdown cells (bottom) 120 h after siRNA transfection, after staining with annexin V (FL4-A channel) and propidium iodide (FL3-A channel). (Left) Gating of all cells (brown); right, distribution of necrotic (bottom right quadrant), early apoptotic (top right quadrant), and apoptotic (top left quadrant) cells along with the respective quantification. (Right) Western blot using antibodies specific to pICln to validate knockdown in cells used for FACS analysis, with tubulin as loading control. (B) Bar graphs representing normalized log₂ fold change in expression of UsnRNP-related proteins and proteins of the SMN complex compared between control siRNA and sipICln cells during pSILAC experiment. Dark- and light-gray bars represent replicates 1 and 2, respectively. (C) Normalized values of fold change in protein expression during pICln depletion from two independent pSILAC experiments. Circles indicate proteins involved in UsnRNP pathway that are listed in the bar graph in B. Red dots in the top right quadrant indicate pICln and SmD1/D2/D3; arrow in the center indicates the other Sm proteins (gray area). Red, blue, and gray colors indicate high, moderate, or insignificant changes in expression, respectively. (D) Western blot analysis of soluble cell lysates prepared for pSILAC, from control and pICln knockdown cells, using anti-pICln, anti–sDMA-Sm (Y12), anti-SmD3, anti-SmD1, and anti-SmF antibodies, respectively. Anti-tubulin antibody was used to monitor sample loading. (E) Quantification of the Western blot images shown in D and pSILAC shown in B and C, from two independent biological replicates. Black, gray, and checkered gray bars indicate control, pSILAC, and Western blot quantifications, respectively.

Figure 5.

Absence of transcriptional or translational regulation of Sm transcripts during pICln knockdown. (A) Normalized fold change of Sm encoding transcripts, G5, LSm2, 10, U1, U2 snRNAs, and SNUP1 in control (black bar) and pICln depletion (gray bars), as analyzed using qRT-PCR experiments, with GAPDH as control for normalization. Error bars indicate SE for three biological replicates (n = 3); **, P < 0.0005. (B, i) Absorbance profile of polysome gradient fractions of control (blue) and pICln knockdown (red) lysates at 260 nm, harvested using Biocomp gradient fractionator. (B, ii) Quantification (top) of control GAPDH mRNA association with polysomes in control (blue) and pICln knockdown (red) cells by RT-PCR analysis, based on the ethidium bromide–stained agarose gel images shown at bottom. (B, iii–vi) RT-PCR analysis of polysome associated SmB/B′ (iii), SmD1 (iv), SmD2 (v), and SmD3 (vi) encoding mRNAs in control (blue) and pICln-deficient (red) cells, with quantification (top) of the ethidium bromide–stained agarose gel images shown at bottom. (C) Western blot analysis showing knockdown of pICln, with tubulin as loading control for the samples used in qRT-PCR experiments (A). (D) Western blot analysis of pICln, SmD1, and SmD3, with tubulin as loading control in input extracts used for polysome gradients (B).

Figure 6.

Sm proteins are degraded by autophagy upon pICln knockdown. (A) Western blot analysis of total cell lysates using antibodies against pICln (knockdown control), β-catenin (control for proteasome inhibition using MG-132), and LC3-II (control for autophagy inhibition using chloroquine). Anti-tubulin antibodies were used to monitor equal loading of all samples. (B and C, top) Western blot analysis of SmD1 (B) and SmD3 (C) using anti-tubulin antibodies to monitor equal loading, in control and pICln knockdown cells upon treatment with water (ddH₂O), ethanol (EtOH)), chloroquine, or MG-132. (Bottom) Quantification of the Western blot from three independent biological experiments (n = 3). Error bars represent SE; *, P < 0.05. Black and gray bars represent control and pICln knockdown conditions, respectively. (D) Western blot analysis of total cell lysates using antibodies against LC3-II (control for autophagy inhibition using bafilomycin A) and pICln, with tubulin as loading control, in control and pICln knockdown conditions. (E) Western blot analysis of total cell lysates using antibodies against SmD1 and D3, with tubulin as loading control, in control and pICln knockdown conditions, upon bafilomycin A treatment, with DMSO as solvent control. (F) Quantification of the Western blot shown in E, from independent biological triplicates (n = 3). Error bars represent SE; *, P < 0.05. Black and gray bars represent control and pICln knockdown conditions, respectively.

Figure 7.

Perturbation of Sm protein homeostasis leads to aggregation in the absence of pICln. (A) Overexpression of SmD3-FLAG in control and pICln-deficient cells analyzed by confocal microscopy by indirect immunofluorescence using antibodies against SMN (green), FLAG (exogenous D3-FLAG; red), or DAPI (blue) upon water (ddH₂O) or chloroquine treatment. White arrowheads indicate regions shown in insets at the top left corner of each image. (B) Confocal microscopy of indirect immunofluorescence of m₃G/m⁷G capped RNA (H20 antibody; green), endogenous SmD3 (red), and DAPI (blue) during control or pICln knockdown, upon chloroquine or water treatment. White arrowheads indicate predominant colocalization/staining pattern observed in splicing speckles or Cajal bodies (CBs; control) or disperse higher-order structures (pICln knockdown). (C) Overexpression of SmD1-FLAG in control and pICln-deficient cells analyzed by confocal microscopy. Indirect immunofluorescence using antibodies targeting SMN (green), FLAG (exogenous D1-FLAG; red), and DAPI (blue) upon water (ddH₂O) or chloroquine treatment. White arrowheads indicate regions shown in insets at the top left corner of each image. (D) Indirect immunofluorescence of endogenous SMN (green) and SmD3 (red) upon treatment of control (i and ii) and pICln-deficient (iii and iv) cells with DMSO solvent or the lysosome inhibitor bafilomycin A. DNA is visualized using DAPI. White arrowheads indicate regions shown in insets at the top left corner of each image. Bars: (A–D, main) 10 µm; (A, C, and D, insets) 1 µm.

Figure 8.

Sm proteins rescued from autophagy form insoluble aggregates. (A) Western blot analysis of lysates from HeLa cells treated with DMSO, compared with soluble extracts prepared from control and pICln siRNA–transfected cells, treated with autophagy inhibitor bafilomycin A, using antibodies specific to pICln and LC3 to monitor pICln knockdown and autophagy inhibition, with tubulin as loading control. (B) Western blot analysis of soluble (supernatant) and insoluble (pellet) fractions of control and pICln knockdown cells upon bafilomycin A treatment. Right top, amido black staining of histones after PVDF membrane transfer as loading control for the insoluble fraction; left top, tubulin (loading control for soluble fraction). (C) Quantification of the Western blots shown in B from n = 1 replicate.

Figure 9.

Elaborate regulation of Sm proteins during UsnRNP assembly prevents cellular proteotoxicity. Schematic representation of the regulatory events ensuring homeostasis of UsnRNPs. For details, see Discussion.