Biochemical and genetic evidence for a role of IGHMBP2 in the translational machinery - PubMed (original) (raw)

. 2009 Jun 15;18(12):2115-26.

doi: 10.1093/hmg/ddp134. Epub 2009 Mar 19.

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Biochemical and genetic evidence for a role of IGHMBP2 in the translational machinery

Mariàngels de Planell-Saguer et al. Hum Mol Genet. 2009.

Abstract

The human motor neuron degenerative disease spinal muscular atrophy with respiratory distress type 1 (SMARD1) is caused by loss of function mutations of immunoglobulin mu-binding protein 2 (IGHMBP2), a protein of unknown function that contains DNA/RNA helicase and nucleic acid-binding domains. Reduced IGHMBP2 protein levels in neuromuscular degeneration (nmd) mice, the mouse model of SMARD1, lead to motor neuron degeneration. We report the biochemical characterization of IGHMBP2 and the isolation of a modifier locus that rescues the phenotype and motor neuron degeneration of nmd mice. We find that a 166 kb BAC transgene derived from CAST/EiJ mice and containing tRNA genes and activator of basal transcription 1 (Abt1), a protein-coding gene that is required for ribosome biogenesis, contains the genetic modifier responsible for motor neuron rescue. Our biochemical investigations show that IGHMBP2 associates physically with tRNAs and in particular with tRNA(Tyr), which are present in the modifier and with the ABT1 protein. We find that transcription factor IIIC-220 kDa (TFIIIC220), an essential factor required for tRNA transcription, and the helicases Reptin and Pontin, which function in transcription and in ribosome biogenesis, are also part of IGHMBP2-containing complexes. Our findings strongly suggest that IGHMBP2 is a component of the translational machinery and that these components can be manipulated genetically to suppress motor neuron degeneration.

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Figures

Figure 1.

Figure 1.

Properties of the monoclonal antibody against IGHMBP2. (A) Total cell lysates from mouse MN1 cells or human 293T cells were resolved on 4–12% NuPAGE gel, blotted and probed with 11-24 mAb. Molecular mass markers (in kilodaltons) are shown on the right. (B) Indirect immunofluorescence of IGHMBP2 on NT2 cell line using 11-24 mAb analyzed by epifluorescence microscopy. (C) FLAG-IGHMBP2 was _in vitro_-translated in the presence of 35S-methionine, and radiolabeled FLAG-IGHMBP2 was immunoprecipitated with non-immune mouse serum (NMS), immune serum from a mouse immunized with recombinant IGHMBP2 (IS-2), the 11-24 mAb, or anti-FLAG (M2) mAb. Immunoprecipitated proteins were resolved on 4–12% NuPAGE gel and detected by autoradiography. Total (T) contains 20% of input used for IPs.

Figure 2.

Figure 2.

IGHMBP2 self-associates and associates with TFIIIC220, and the helicases Reptin and Pontin. (A) Identification of IGHMBP2-associated proteins. Immunoprecipitations (IPs) were performed with anti-FLAG or 11-24 (anti-IGHMBP2) antibodies from 293 cells expressing FLAG-IGHMBP2 or from mock-transfected 293 cells. Immunoprecipitates from the anti-FLAG resins were eluted with 3× FLAG peptide. Immunoprecipitates from the 11-24 IP (beads) and eluates from the anti-FLAG IPs were resolved on a 4–12% NuPAGE gel and visualized by Coomassie blue staining. Arrow indicates the FLAG-IGHMBP2 protein. Proteins that co-precipitate with both antibodies (band P50 is obscured from the 11-24 IPs from the antibody chain) are indicated. Molecular mass markers (in kilodaltons) are shown on the right. The heavy and light chains of the 11-24 antibody are indicated. Protein bands P100, P75, P65 and P50 were excised from the gel (FLAG-IGHMBP2 eluate) and identified by mass spectrometry. The corresponding proteins (and Genbank protein IDs) from these bands are listed below. (B–D) Validation of IGHMBP2-interacting proteins by reverse co-IP experiments. (B) Two hundred and ninety-three cells expressing myc-IGHMBP2 were transfected with FLAG-Reptin (lane 2) or with empty vector (mock, lane 1). IPs were performed with anti-FLAG antibody from the mock-transfected (lane 3) or FLAG-Reptin-transfected cells (lane 4), and the immunoprecipitates were probed with anti-myc (9E10) antibody on western blot. (C) Two hundred and ninety-three cells expressing myc-IGHMBP2 were transfected with FLAG-Pontin (lane 2) or with empty vector (mock, lane 1). IPs were performed with anti-FLAG antibody from the mock-transfected (lane 3) or FLAG-Pontin-transfected cells (lane 4), and the immunoprecipitates were probed with anti-myc (9E10) antibody on western blot. (D) IPs were performed from 293 cells using antibodies against: TFIIIC220 (lane 5), α-tubulin (lane 4), IGHMBP2 (11-24 mAb, lane 3), NMS (non-immune mouse serum; negative control, lane 2). The immunoprecipitates were probed with 11-24 (anti-IGHMBP2 mAb) on western blot. (E) IGHMBP2 self-associates. 293T cells were transfected with FLAG-IGHMBP2 (F), or myc-IGHMBP2 (M), or both FLAG-IGHMBP2 and myc-IGHMBP2 (F/M), or were mock-transfected (C). IPs were performed with either anti-FLAG or anti-myc antibodies, and the immunoprecipitates were probed on western blots with anti-myc (top panel) or anti-FLAG (bottom panel) antibodies. Total represents ∼10% of lysate prior to IPs. Arrow indicates epitope-tagged IGHMBP2 protein.

Figure 3.

Figure 3.

IGHMBP2 associates with small RNAs and in particular with tRNA-Tyr. (A) Lysates from mouse MN1 cells stably expressing FLAG-IGHMBP2 or vector only (FLAG-vector) were probed on a western blot with a monoclonal antibody against the FLAG epitope. (B) RNA immunoprecipitations (RNA-IP) were performed with anti-FLAG antibody from FLAG–IGHMBP2-expressing or mock-transfected (vector only) MN1 cells. RNA was isolated from the immunoprecipitates, 3′-end-labeled with [5′-32P]-pCp and T4 RNA ligase and resolved by electrophoresis on a 10% denaturing PAGE. Nucleotide sizes of the radiolabeled marker (M) are shown on the left. Red arrow at ∼40 nt indicates small RNAs that were directionally cloned and sequenced. (C) Secondary structure of mouse tRNA-Tyr (tRNA-882; numbering is adapted from 13) with cloned sequences shown in red, including the post-transcriptionally added CCA trinucleotide at the 3′-end. Nucleotide differences of tRNA 880 (tRNA-Tyr2) are indicated in green; all other nucleotides between the various tRNAs-Tyr listed above are identical. The pseudo-uridine modification of uridine present in the center of the anticodon of all tRNA-Tyr is shown with the symbol ψ. (D) Sequences of mouse tRNA-Tyr genes contained in the modifier locus (see Fig. 4). Introns are in lower case; note that intronic sequences differ between the various tRNA-Tyr genes. (E) RNA-IP was performed with 11-24 mAb against endogenous IGHMBP2 from human HeLa cells or with non-immune mouse serum (NMS, negative control). RNA was isolated from the immunoprecipitates, 3′-end-labeled with [5′-32P]-pCp and T4 RNA ligase and resolved by electrophoresis on a 10% denaturing PAGE. Nucleotide sizes of the radiolabeled marker (M) are shown on the left. Red arrow indicates tRNAs. (F) RNA-IP was performed from HeLa cells as in (E), and the isolated RNA was transferred to a nylon membrane and probed with a 5′-end-labeled DNA oligo, antisense to tRNA-Tyr.

Figure 4.

Figure 4.

MnM modifier locus and BAC-27K3 rescuing transgene. (A) Transgenic positional complementation cloning the Mnm modifier. Schematic of the 430 kb Mnm modifier locus and of the BAC 27k3 (blue) that suppresses nmd motor neuron degeneration and contains the sequences necessary to confer the Mnm effect. The 18.1 kb _Eco_RI fragment from BAC-27k3 that contains the five tRNA-Tyr and Abt1 genes is shown magnified below. The polymorphisms between B6 and CAST are also shown. Boxed genes and exons above the line are transcribed from left to right and those below the line are transcribed from right to left. Coding genes and functional tRNAs are depicted as is a non-coding EST (ncEST). (B) Cross-sections of the motor branch of the femoral nerve from a 35-day-old B6-+/+ mouse, a 41-day-old B6-nmd/nmd mouse and a 37-day-old B6-Tg27k3 nmd/nmd rescued mouse. There is a significant reduction in the total number of myelinated axons in the nmd femoral motor nerves that is completely rescued by the 27k3 BAC transgene. (C) Axon counts from femoral motor nerves from homozygous B6 nmd mice (nmd/nmd, n = 13), wild-type B6 mice (+/+, n = 6), B6-Tg27k3 nmd/nmd rescued mice (Tg nmd/nmd, n = 8) and B6-Tg27k3 (Tg +/+, n = 4).

Figure 5.

Figure 5.

The genetic modifier rescues the motor neuron degeneration of nmd mice without restoring the protein levels of IGHMBP2. (A) RT–PCR was performed with primers spanning the point mutation in intron 4 of Ighmbp2 gene of nmd mice that leads to the inclusion of 23 nucleotides in the aberrant Ighmbp2 pre-mRNA from nmd mice. The aberrant Ighmbp2 transcript is the major transcript generated in nmd mice and in the B6-Tg(BAC27k3) nmd mice. (B) Indicated amounts of brain tissue lysates from wild-type (wt), nmd or B6-Tg(BAC27k3) nmd mice were probed with the indicated antibodies on western blots.

Figure 6.

Figure 6.

IGHMBP2 associates with ABT1. 293T cells were co-transfected with FLAG-IGHMBP2 and myc-ABT1. IPs were performed with anti-myc antibody (left panel) or with anti-FLAG antibody (right panel), and the immunoprecipitates were probed on western blots with anti-FLAG antibody (to detect FLAG-IGHMBP2) and with 9E10 anti-myc antibody (to detect myc-ABT1). Total represents 10% of lysate prior to IP.

References

    1. Grohmann K., Schuelke M., Diers A., Hoffmann K., Lucke B., Adams C., Bertini E., Leonhardt-Horti H., Muntoni F., Ouvrier R., et al. Mutations in the gene encoding immunoglobulin mu-binding protein 2 cause spinal muscular atrophy with respiratory distress type 1. Nat. Genet. 2001;29:75–77. - PubMed
    1. Grohmann K., Varon R., Stolz P., Schuelke M., Janetzki C., Bertini E., Bushby K., Muntoni F., Ouvrier R., Van Maldergem L., et al. Infantile spinal muscular atrophy with respiratory distress type 1 (SMARD1) Ann. Neurol. 2003;54:719–724. - PubMed
    1. Grohmann K., Rossoll W., Kobsar I., Holtmann B., Jablonka S., Wessig C., Stoltenburg-Didinger G., Fischer U., Hubner C., Martini R., et al. Characterization of Ighmbp2 in motor neurons and implications for the pathomechanism in a mouse model of human spinal muscular atrophy with respiratory distress type 1 (SMARD1) Hum. Mol. Genet. 2004;13:2031–2042. - PubMed
    1. Fukita Y., Mizuta T.R., Shirozu M., Ozawa K., Shimizu A., Honjo T. The human S mu bp-2, a DNA-binding protein specific to the single-stranded guanine-rich sequence related to the immunoglobulin mu chain switch region. J. Biol. Chem. 1993;268:17463–17470. - PubMed
    1. Kerr D., Khalili K. A recombinant cDNA derived from human brain encodes a DNA binding protein that stimulates transcription of the human neurotropic virus JCV. J. Biol. Chem. 1991;266:15876–15881. - PubMed

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