Nuclear poly(A) binding protein 1 (PABPN1) and Matrin3 interact in muscle cells and regulate RNA processing

Abstract

The polyadenylate binding protein 1 (PABPN1) is a ubiquitously expressed RNA binding protein vital for multiple steps in RNA metabolism. Although PABPN1 plays a critical role in the regulation of RNA processing, mutation of the gene encoding this ubiquitously expressed RNA binding protein causes a specific form of muscular dystrophy termed oculopharyngeal muscular dystrophy (OPMD). Despite the tissue-specific pathology that occurs in this disease, only recently have studies of PABPN1 begun to explore the role of this protein in skeletal muscle. We have used co-immunoprecipitation and mass spectrometry to identify proteins that interact with PABPN1 in mouse skeletal muscles. Among the interacting proteins we identified Matrin 3 (MATR3) as a novel protein interactor of PABPN1. The MATR3 gene is mutated in a form of distal myopathy and amyotrophic lateral sclerosis (ALS). We demonstrate, that like PABPN1, MATR3 is critical for myogenesis. Furthermore, MATR3 controls critical aspects of RNA processing including alternative polyadenylation and intron retention. We provide evidence that MATR3 also binds and regulates the levels of long non-coding RNA (lncRNA) Neat1 and together with PABPN1 is required for normal paraspeckle function. We demonstrate that PABPN1 and MATR3 are required for paraspeckles, as well as for adenosine to inosine (A to I) RNA editing of Ctn RNA in muscle cells. We provide a functional link between PABPN1 and MATR3 through regulation of a common lncRNA target with downstream impact on paraspeckle morphology and function. We extend our analysis to a mouse model of OPMD and demonstrate altered paraspeckle morphology in the presence of endogenous levels of alanine-expanded PABPN1. In this study, we report protein-binding partners of PABPN1, which could provide insight into novel functions of PABPN1 in skeletal muscle and identify proteins that could be sequestered with alanine-expanded PABPN1 in the nuclear aggregates found in OPMD.

Figure 1.

PABPN1 is present in multiple protein complexes. (A) Whole cell lysate from control myotubes or myotubes overexpressing PABPN1 (PABPN1 O/E) were resolved by blue native polyacrylamide gel electrophoresis (BN-PAGE) (top) and SDS-PAGE (bottom) and analyzed by immunoblotting with anti-PABPN1 antibody (9). Higher molecular weight bands potentially representing PABPN1 protein complexes are indicated by arrowheads. HSP90 SERVES as a loading control. (B) Whole cell lysate from myotubes overexpressing PABPN1 was resolved by BN-PAGE in the first dimension followed by SDS-PAGE in the second dimension. Immunoblotting was performed with anti-PABPN1 antibody to confirm the presence of PABPN1 in higher molecular weight bands (indicated by arrowheads). (C) Whole cell lysates from myoblasts (top) and myotubes (bottom) were analyzed by sedimentation through 10–30% glycerol gradients. Alternate gradient fractions from top to bottom were loaded and resolved by SDS-PAGE. Input was also loaded as a control. The presence of PABPN1 in the glycerol gradient fractions was detected by immunoblotting with an anti-PABPN1 antibody. The migration of PABPN1 in heavier fractions in lysates from myotubes is indicated by the box. (D) Whole cell lysate from primary myoblasts and differentiated myotubes were analyzed by sedimentation through 10–30% glycerol gradients. Alternate gradient fractions from top to bottom were loaded from left to right and resolved by Blue Native-PAGE (the sample representing fraction 19 was lost due to a technical issue). Immunoblotting with a PABPN1 antibody detects PABPN1 in a major band of ∼66 kDa and some fainter higher bands in fractions collected from myoblasts. PABPN1 appears in a band of higher molecular weight (∼480 kDa) in the heavier glycerol gradient fractions collected from myotubes (boxed in black).

Figure 2.

PABPN1 interacts with multiple proteins in skeletal muscle lysate. (A) Lysates collected from the indicated tissues were resolved by SDS-PAGE and analyzed by immunoblotting for PABPN1. Histone H3 serves as a loading control. A higher exposure of the PABPN1 immunoblot is shown to visualize PABPN1 in the skeletal muscle lysate. (B) Lysates obtained from control gastrocnemius skeletal muscle and gastrocnemius skeletal muscle transgenically overexpressing PABPN1 (A10.1) were resolved by SDS-PAGE and analyzed by immunoblotting for PABPN1. Histone H3 acts as a loading control. (C) A schematic outlining the experimental approach to identify PABPN1-interacting proteins in mouse muscle is shown. We isolate gastrocnemius muscles and then immunoprecipitate with either a PABPN1 antibody (9) or control IgG, followed by mass spectrometry as described in Material and Methods. (D) Table listing the PABPN1-interacting proteins that co-immunoprecipitate with PABPN1 from murine skeletal muscles is shown. (E) Protein interactions identified by mass spectrometry (PABPC1 and ARC1) were validated by co-immunoprecipitation from murine skeletal muscle lysates. The bound fractions from control (IgG) and PABPN1 immunoprecipitations (IP) were resolved by SDS-PAGE and immunoblotted for PABPN1, PABPC1 and ARC1. The Input is shown as a control. Results shown are typical of three independent experiments.

Figure 3.

PABPN1 interacts with MATR3. (A) Input and bound samples from control (IgG) and PABPN1 immunoprecipitations (IP) from lysates obtained from uninjured muscles or injured muscles 6 days post-injury were resolved by SDS-PAGE and immunoblotted for PABPN1 and MATR3. Results shown are typical of three independent experiments. (B) Primary myoblasts were transfected with a plasmid expressing myc-MATR3 and differentiated into myotubes for 48 h. These transfected myotubes were stained with anti-PABPN1 antibody to detect endogenous PABPN1 (FITC/Green) and anti-myc antibody to detect myc-MATR3 (Texas red). Merged images of FITC and Texas red staining are shown to visualize co-localization (Yellow) in myonuclei. DAPI staining marks the nucleus. Four independent experiments were conducted with similar results. Representative images are shown for two independent myonuclei from independent transfections. (C) Lysates from C2C12 cell line myotubes were analyzed by sedimentation through 10–30% glycerol gradients. Gradient fractions from top to bottom were loaded from left to right and resolved by SDS-PAGE. An Input sample is shown on the right. Immunoblotting was performed to detect PABPN1 and MATR3 in the glycerol gradient fractions. The black box indicates fractions that contain both PABPN1 and MATR3. Immunoblotting was also performed to detect EXOSC10 (RNA exosome complex) and CPSF73 (cleavage and polyadenylation complex) in the fractions. (D) Whole cell lysate from primary myotubes was resolved by BN-PAGE in the first dimension followed by SDS-PAGE in the second dimension. Immunoblotting was performed to detect MATR3 and PABPN1 (indicated by arrowheads) in the same higher molecular weight bands. (E) Lysates from control primary myoblasts or primary myoblasts transfected with a plasmid expressing Flag-PABPN1 were either treated with RNase A (+) or left untreated (–), followed by immunoprecipitation with an anti-Flag antibody. Immunoblotting was performed to detect MATR3 and PABPN1. (F) Direct protein-protein interaction between MATR3 and PABPN1 was assessed by incubating recombinant His-PABPN1 with control GST protein alone or GST-MATR3, followed by a GST-pulldown and immunoblotting for GST and His-PABPN1. Asterisks indicate the positions of two degradation products of GST-MATR3.

Figure 4.

MATR3 binds to both coding and non-coding myogenic transcripts. To test for transcripts associated with MATR3, immunoprecipitations were performed from UV crosslinked primary myoblasts using control IgG antibody or MATR3 antibody. Bound RNAs, both (A) coding (Myog, MyoD, Pitx2, Gapdh, Acta1, Mef2d and Tmod1) and (B) non-coding (Linc-MD1, Malat1 and 7SK), were detected by qRT-PCR. For each transcript analyzed, the bound RNA was normalized to input and graphed as fold enrichment over binding to control IgG antibody, which was set to 1.0. Results are the average of three biological replicates. Error bars represent standard error of the mean (*P < 0.05).

Figure 5.

MATR3 is required for normal myoblast proliferation and differentiation. (A) Primary myoblasts were transfected with Control siRNA or siRNA targeting either PABPN1 or MATR3. Immunoblotting was performed with anti-PABPN1 and anti-MATR3 antibodies to confirm depletion of PABPN1 and MATR3, respectively. Tubulin serves as a loading control. (B) Cells were labeled with EdU and actively proliferating cells were detected by staining with an anti-Edu Alexa Fluor 594 antibody. Hoechst was used to mark the position of the nuclei. (C) The number of EdU positive nuclei and Alexa Fluor 594 signal intensity was quantified using Cell Profiler software. A frequency graph was generated by plotting signal intensity of Edu staining on the X-axis and the number of Edu positive primary myoblasts binned on the Y-axis. (D) Primary myoblasts transfected with Control siRNA or siRNA targeting PABPN1 or MATR3 were differentiated. Protein extracts obtained from primary myotubes were analyzed by immunoblotting for PABPN1, MATR3 and Myogenin. Tubulin serves as a loading control. (E) Phase-contrast microscopy images were captured to monitor in vitro myogenesis. Representative images are shown from Day 1 and Day 2 of differentiation for cells transfected with Control siRNA or siRNA targeting PABPN1 or MATR3.

Figure 6.

MATR3 does not regulate bulk polyadenylation in primary myoblasts. (A) Total RNA isolated from myoblasts transfected with Control siRNA or siRNA targeting PABPN1 or MATR3 was analyzed for bulk poly (A) tail length by resolving 3′-end labeled poly(A) tracts on a denaturing polyacrylamide gel as described in Materials and Methods. (B) Bulk poly (A) tail lengths were quantified using ImageQuant software. Relative intensities of the bulk poly (A) tails on the Y-axis were plotted against increasing poly (A) tail lengths on the X-axis. Approximate size of RNA size markers are indicated by dotted lines.

Figure 7.

Regulation of shared transcripts by MATR3 and PABPN1. (A) Schematic of qRT-PCR strategy with primers specific to the coding sequence (CDS) used to detect Total transcript or the distal 3′ UTR to analyze differential use of the proximal polyadenylation site (PAS1) or the distal polyadenylation site (PAS2) in candidate mRNAs. (B) Primary myoblasts were transfected with control siRNA or siRNA targeting PABPN1 or MATR3. qRT-PCR with primers detecting the CDS (Total) and the long 3′ UTR (distal UTR) was utilized to quantify levels of Tmod1, Timp2, Psme3 and Vldlr RNAs. The levels of the longer 3′ UTR (distal 3′ UTR) are presented relative to the Total mRNA (Total) normalized to Hprt. The experiment was carried out at least five independent times. Error bars represent standard error of the mean (*P < 0.05). (C) qRT-PCR was performed to assess the levels of Mat2a transcript containing the retained intron (Mat2a-RI) and total Mat2a transcript in myoblasts depleted of PABPN1 and MATR3 as compared to control myoblasts. The levels of the Mat2a transcript containing the retained intron (Mat2a-RI) are presented relative to the total mRNA (Mat2a-Total) normalized to Hprt. The experiment was carried out seven independent times. Error bars represent standard error of the mean (*P < 0.05). (D) MAT2A protein levels were assessed in primary myoblasts transfected with Control siRNA or siRNA targeting either PABPN1 or MATR3. Immunoblotting was performed using a MAT2A antibody with GAPDH serving as a loading control. Levels of MAT2A were quantified by densitometry using Fiji imaging software and are presented as fold change relative to samples from control siRNA normalized to GAPDH. The experiment was carried out three independent times. Error bars represent standard error of the mean (*P < 0.05). (E) Levels of S-adenosyl methionine (SAM) were measured in control primary myoblasts and primary myoblasts depleted of PABPN1 or MATR3 as described in Materials and Methods. The experiment was carried out three independent times. Error bars represent standard error of the mean (*P < 0.05).

Figure 8.

PABPN1 and MATR3 regulate nuclear paraspeckle function. (A) Primary myoblasts were transfected with Control siRNA or siRNA targeting PABPN1 or MATR3. qRT-PCR was performed to assess the levels of Neat1 lncRNA. Levels of Neat1 RNA are presented as fold change relative to the control transcript, Gapdh and normalized to Control siRNA. (B) RNA immunoprecipitations were performed from primary myoblasts using control IgG or MATR3 antibody. Bound RNA is normalized to input and is presented as fold enrichment over binding to control IgG antibody, which was set to 1.0. Results are representative of three independent experiments. Error bars represent standard error of the mean (*P < 0.05). (C) Neat1 RNA-FISH was performed using a Cy3 labeled Neat1 probe, to visualize paraspeckles in control myoblasts or myoblasts depleted of PABPN1 or MATR3. Two independent myonuclei are shown. DAPI staining marks the nucleus. (D) Paraspeckles were counted in at least 100 cells from independent fields for each siRNA (Control, PABPN1, MATR3) and graphed as fold-change relative to control siRNA treatment. Experiments were carried out three independent times. Error bars represent standard error of the mean (*P < 0.05). (E) A diagram representing the PCR amplification of an edited section of the Ctn 3′ UTR (69) to analyze adenosine to inosine (A to I) RNA editing. Also shown is a typical electropherogram detecting an RNA editing event (arrow) that is identified as an adenosine (A) to guanosine (G) change in the sequenced PCR product. (F) PCR products amplified from cDNA were cloned and sequenced. The number of sequenced A to I editing events in control myoblasts, myoblasts depleted of PABPN1 or MATR3 and myoblasts with a concomitant depletion of Neat1 RNA (Neat1+PABPN1 and Neat1+MATR3) is presented as the percentage of sequenced clones with at least one RNA editing event (% Editing of Ctn). (G) Whole cell lysates prepared from primary myoblasts transfected with control siRNA or siRNA targeting PABPN1 or MATR3 were analyzed by sedimentation through 10–30% glycerol gradients. Gradient fractions from top to bottom were loaded left to right together with an ‘Input’ lane and resolved by SDS-PAGE, followed by immunoblotting for the paraspeckle component SFPQ. (H) Skeletal muscle lysate obtained from wildtype mice (Ala10/Ala10) or mice carrying one wildtype and one mutant allele (Ala10/Ala17) of PABPN1 was analyzed by sedimentation through 10–30% glycerol gradients. Glycerol gradient fractions 1 to 6 (top to bottom) were loaded left to right and resolved by SDS-PAGE followed by immunoblotting for SFPQ.

Figure 9.

The requirement for PABPN1 in paraspeckle function is conserved in human myoblasts. (A) Immunofluorescence was performed to detect PABPN1 protein in control human myoblasts and PABPN1-KO human myoblasts created with CRISPR-Cas9 as described in Materials and Methods. DAPI staining marks the position of the nucleus. (B) Immunoblot analysis was performed to analyze levels of PABPN1 protein in PABPN1-KO human myoblasts as compared to control human myoblasts. Histone H3 serves as a loading control. (C) Myoblasts were fractionated to determine nuclear-cytoplasmic localization of candidate transcripts. Efficient nucleocytoplasmic fractionation was confirmed by immunoblotting with Histone H3 and GAPDH antibodies to mark the nuclear (Nuc) and cytoplasmic (Cyt) fractions, respectively. (D) The steady-state distribution of known paraspeckle-mediated nuclear retained transcripts (NUP43 and PAICS) was established by performing qRT-PCR using transcript specific primers on the nuclear (Nuc) and cytoplasmic (Cyt) fractions prepared from control and PABPN1-KO human myoblasts. NUP43 and PAICS transcripts were normalized to the endogenous transcript RPLP0 and are presented here as a percentage of total transcript Transcript distribution as a fraction of total). Experiments were performed four independent times. Error bars represent standard error of the mean (*P < 0.05).