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Identification and characterization of RBM44 as a novel intercellular bridge protein.

Iwamori T, Lin YN, Ma L, Iwamori N, Matzuk MM - PLoS ONE (2011)

Bottom Line: RBM44 is expressed most highly in pachytene and secondary spermatocytes, but disappears abruptly in spermatids.We discovered that RBM44 interacts with itself and TEX14 using yeast two-hybrid, mammalian two-hybrid, and immunoprecipitation.To define the in vivo function of RBM44, we generated a targeted deletion of Rbm44 in mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
Intercellular bridges are evolutionarily conserved structures that connect differentiating germ cells. We previously reported the identification of TEX14 as the first essential intercellular bridge protein, the demonstration that intercellular bridges are required for male fertility, and the finding that intercellular bridges utilize components of the cytokinesis machinery to form. Herein, we report the identification of RNA binding motif protein 44 (RBM44) as a novel germ cell intercellular bridge protein. RBM44 was identified by proteomic analysis after intercellular bridge enrichment using TEX14 as a marker protein. RBM44 is highly conserved between mouse and human and contains an RNA recognition motif of unknown function. RBM44 mRNA is enriched in testis, and immunofluorescence confirms that RBM44 is an intercellular bridge component. However, RBM44 only partially localizes to TEX14-positive intercellular bridges. RBM44 is expressed most highly in pachytene and secondary spermatocytes, but disappears abruptly in spermatids. We discovered that RBM44 interacts with itself and TEX14 using yeast two-hybrid, mammalian two-hybrid, and immunoprecipitation. To define the in vivo function of RBM44, we generated a targeted deletion of Rbm44 in mice. Rbm44 male mice produce somewhat increased sperm, and show enhanced fertility of unknown etiology. Thus, although RBM44 localizes to intercellular bridges during meiosis, RBM44 is not required for fertility in contrast to TEX14.

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RBM44 targeted deletion in vivo.A, The strategy and conditional targeting vector to mutate the Rbm44 locus in ES cells and delete the RRM domain in mice: open white rectangle, PGK1-neomycin expression cassette; black arrowhead, frt sequence; open white arrowhead, loxP sequence; B, BglII site; S, SacI site. Exons 11 and 12 are deleted in Rbm44  mice. Southern probes are shown as black boxes. B, The mating strategy to generate a ubiquitous deletion of RBM44. C, The strategy to detect Rbm44 WT, targeted, and deleted alleles. D, Southern blot analysis using restriction enzyme SacI and 3′probe shows recombination of Rbm44 in targeted ES cells (wild-type allele, 12.8 kb; targeted Rbm44frt-neo-frt–loxP allele, 9.7 kb). E, Southern blot analysis using restriction enzyme BglII and 5′probe shows targeted deletion of Rbm44 in mice (wild-type allele, 9.8 kb; targeted Rbm44frt-neo-frt-loxP allele, 7.2 kb; Rbm44  allele, 4.2 kb). F, PCR genotyping of Rbm44  mice. (WT, 313 bp; WT in bottom panel, 1636 bp; , 403 bp). G, Western blot analyses of total testis extracts of wild-type (WT), Rbm44 heterozygous (Rbm44+/−), and  (Rbm44−/−) mice using anti-RBM44 antibody generated against amino acids 471–609 (N-terminal of the deletion) and anti-ACTIN antibody for a control.
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pone-0017066-g008: RBM44 targeted deletion in vivo.A, The strategy and conditional targeting vector to mutate the Rbm44 locus in ES cells and delete the RRM domain in mice: open white rectangle, PGK1-neomycin expression cassette; black arrowhead, frt sequence; open white arrowhead, loxP sequence; B, BglII site; S, SacI site. Exons 11 and 12 are deleted in Rbm44 mice. Southern probes are shown as black boxes. B, The mating strategy to generate a ubiquitous deletion of RBM44. C, The strategy to detect Rbm44 WT, targeted, and deleted alleles. D, Southern blot analysis using restriction enzyme SacI and 3′probe shows recombination of Rbm44 in targeted ES cells (wild-type allele, 12.8 kb; targeted Rbm44frt-neo-frt–loxP allele, 9.7 kb). E, Southern blot analysis using restriction enzyme BglII and 5′probe shows targeted deletion of Rbm44 in mice (wild-type allele, 9.8 kb; targeted Rbm44frt-neo-frt-loxP allele, 7.2 kb; Rbm44 allele, 4.2 kb). F, PCR genotyping of Rbm44 mice. (WT, 313 bp; WT in bottom panel, 1636 bp; , 403 bp). G, Western blot analyses of total testis extracts of wild-type (WT), Rbm44 heterozygous (Rbm44+/−), and (Rbm44−/−) mice using anti-RBM44 antibody generated against amino acids 471–609 (N-terminal of the deletion) and anti-ACTIN antibody for a control.

Mentions: To determine the spatiotemporal expression of RBM44, we generated antibodies to independent regions of RBM44. One antibody was generated against N-terminal amino acids 471–609 and the other antibody to C-terminal amino acids 740–1013 of the mouse RBM44 protein that contained the RRM domain (amino acids 793–860). Both peptides were used to generate polyclonal antibodies in guinea pigs using methods described previously [9]. The antibodies were affinity purified with the RBM44 antigens using the AminoLink Plus Immobilization Kit (Pierce, Rockford, IL). The antibody to amino acids 740–1013 of mouse RBM44 was used in Figures 1, 2, 3, 4, and 5. The antibody to N-terminal amino acids 740–1013 of mouse RBM44 was used in Figures 8 and 9 to confirm that our mutation generated a mutation.


Identification and characterization of RBM44 as a novel intercellular bridge protein.

Iwamori T, Lin YN, Ma L, Iwamori N, Matzuk MM - PLoS ONE (2011)

RBM44 targeted deletion in vivo.A, The strategy and conditional targeting vector to mutate the Rbm44 locus in ES cells and delete the RRM domain in mice: open white rectangle, PGK1-neomycin expression cassette; black arrowhead, frt sequence; open white arrowhead, loxP sequence; B, BglII site; S, SacI site. Exons 11 and 12 are deleted in Rbm44  mice. Southern probes are shown as black boxes. B, The mating strategy to generate a ubiquitous deletion of RBM44. C, The strategy to detect Rbm44 WT, targeted, and deleted alleles. D, Southern blot analysis using restriction enzyme SacI and 3′probe shows recombination of Rbm44 in targeted ES cells (wild-type allele, 12.8 kb; targeted Rbm44frt-neo-frt–loxP allele, 9.7 kb). E, Southern blot analysis using restriction enzyme BglII and 5′probe shows targeted deletion of Rbm44 in mice (wild-type allele, 9.8 kb; targeted Rbm44frt-neo-frt-loxP allele, 7.2 kb; Rbm44  allele, 4.2 kb). F, PCR genotyping of Rbm44  mice. (WT, 313 bp; WT in bottom panel, 1636 bp; , 403 bp). G, Western blot analyses of total testis extracts of wild-type (WT), Rbm44 heterozygous (Rbm44+/−), and  (Rbm44−/−) mice using anti-RBM44 antibody generated against amino acids 471–609 (N-terminal of the deletion) and anti-ACTIN antibody for a control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045441&req=5

pone-0017066-g008: RBM44 targeted deletion in vivo.A, The strategy and conditional targeting vector to mutate the Rbm44 locus in ES cells and delete the RRM domain in mice: open white rectangle, PGK1-neomycin expression cassette; black arrowhead, frt sequence; open white arrowhead, loxP sequence; B, BglII site; S, SacI site. Exons 11 and 12 are deleted in Rbm44 mice. Southern probes are shown as black boxes. B, The mating strategy to generate a ubiquitous deletion of RBM44. C, The strategy to detect Rbm44 WT, targeted, and deleted alleles. D, Southern blot analysis using restriction enzyme SacI and 3′probe shows recombination of Rbm44 in targeted ES cells (wild-type allele, 12.8 kb; targeted Rbm44frt-neo-frt–loxP allele, 9.7 kb). E, Southern blot analysis using restriction enzyme BglII and 5′probe shows targeted deletion of Rbm44 in mice (wild-type allele, 9.8 kb; targeted Rbm44frt-neo-frt-loxP allele, 7.2 kb; Rbm44 allele, 4.2 kb). F, PCR genotyping of Rbm44 mice. (WT, 313 bp; WT in bottom panel, 1636 bp; , 403 bp). G, Western blot analyses of total testis extracts of wild-type (WT), Rbm44 heterozygous (Rbm44+/−), and (Rbm44−/−) mice using anti-RBM44 antibody generated against amino acids 471–609 (N-terminal of the deletion) and anti-ACTIN antibody for a control.
Mentions: To determine the spatiotemporal expression of RBM44, we generated antibodies to independent regions of RBM44. One antibody was generated against N-terminal amino acids 471–609 and the other antibody to C-terminal amino acids 740–1013 of the mouse RBM44 protein that contained the RRM domain (amino acids 793–860). Both peptides were used to generate polyclonal antibodies in guinea pigs using methods described previously [9]. The antibodies were affinity purified with the RBM44 antigens using the AminoLink Plus Immobilization Kit (Pierce, Rockford, IL). The antibody to amino acids 740–1013 of mouse RBM44 was used in Figures 1, 2, 3, 4, and 5. The antibody to N-terminal amino acids 740–1013 of mouse RBM44 was used in Figures 8 and 9 to confirm that our mutation generated a mutation.

Bottom Line: RBM44 is expressed most highly in pachytene and secondary spermatocytes, but disappears abruptly in spermatids.We discovered that RBM44 interacts with itself and TEX14 using yeast two-hybrid, mammalian two-hybrid, and immunoprecipitation.To define the in vivo function of RBM44, we generated a targeted deletion of Rbm44 in mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
Intercellular bridges are evolutionarily conserved structures that connect differentiating germ cells. We previously reported the identification of TEX14 as the first essential intercellular bridge protein, the demonstration that intercellular bridges are required for male fertility, and the finding that intercellular bridges utilize components of the cytokinesis machinery to form. Herein, we report the identification of RNA binding motif protein 44 (RBM44) as a novel germ cell intercellular bridge protein. RBM44 was identified by proteomic analysis after intercellular bridge enrichment using TEX14 as a marker protein. RBM44 is highly conserved between mouse and human and contains an RNA recognition motif of unknown function. RBM44 mRNA is enriched in testis, and immunofluorescence confirms that RBM44 is an intercellular bridge component. However, RBM44 only partially localizes to TEX14-positive intercellular bridges. RBM44 is expressed most highly in pachytene and secondary spermatocytes, but disappears abruptly in spermatids. We discovered that RBM44 interacts with itself and TEX14 using yeast two-hybrid, mammalian two-hybrid, and immunoprecipitation. To define the in vivo function of RBM44, we generated a targeted deletion of Rbm44 in mice. Rbm44 male mice produce somewhat increased sperm, and show enhanced fertility of unknown etiology. Thus, although RBM44 localizes to intercellular bridges during meiosis, RBM44 is not required for fertility in contrast to TEX14.

Show MeSH
Related in: MedlinePlus