Dense cataract and microphthalmia (dcm) in BALB/c mice is caused by mutations in the GJA8 locus

A spontaneous mutation in BALB/c mice that causes congenital dense cataract and microphthalmia (dcm) was reported previously. This abnormality was found to be inheritable and the mode of inheritance indicated that this phenotype is due to mutation of an autosomal recessive gene. We performed genetic screen to identify the underlying mutations through linkage analysis with the dcm progenies of F1 intercross. We identified the region of mutation on chromosome 3 and further mapping and sequence analysis identified the mutation in the GJA8 gene that encodes for connexin 50. The mutation represents a single nucleotide change at position 64 (G to C) that results in a change in the amino acid glycine to arginine at position 22 (G22R) and is identical to the mutation previously characterized as lop10. However, the phenotype of these mice differ from that of lop10 mice and since it is one of the very few genetic models with recessive pattern of inheritance, we propose that dcm mice can serve as a useful model for studying the dynamics and interaction of the gap junction formation in mouse eye development

Eggs of the mosquito Aedes aegypti survive desiccation by rewiring their polyamine and lipid metabolism.

Upon water loss, some organisms pause their life cycles and escape death. While widespread in microbes, this is less common in animals. Aedes mosquitoes are vectors for viral diseases. Aedes eggs can survive dry environments, but molecular and cellular principles enabling egg survival through desiccation remain unknown. In this report, we find that Aedes aegypti eggs, in contrast to Anopheles stephensi, survive desiccation by acquiring desiccation tolerance at a late developmental stage. We uncover unique proteome and metabolic state changes in Aedes embryos during desiccation that reflect reduced central carbon metabolism, rewiring towards polyamine production, and enhanced lipid utilisation for energy and polyamine synthesis. Using inhibitors targeting these processes in blood-fed mosquitoes that lay eggs, we infer a two-step process of desiccation tolerance in Aedes eggs. The metabolic rewiring towards lipid breakdown and dependent polyamine accumulation confers resistance to desiccation. Furthermore, rapid lipid breakdown is required to fuel energetic requirements upon water reentry to enable larval hatching and survival upon rehydration. This study is fundamental to understanding Aedes embryo survival and in controlling the spread of these mosquitoes

The Cytoplasmic Capping Complex Assembles on Adapter Protein Nck1 Bound to the Proline-Rich C-Terminus of Mammalian Capping Enzyme

<div><p>Cytoplasmic capping is catalyzed by a complex that contains capping enzyme (CE) and a kinase that converts RNA with a 5′-monophosphate end to a 5′ diphosphate for subsequent addition of guanylic acid (GMP). We identify the proline-rich C-terminus as a new domain of CE that is required for its participation in cytoplasmic capping, and show the cytoplasmic capping complex assembles on Nck1, an adapter protein with functions in translation and tyrosine kinase signaling. Binding is specific to Nck1 and is independent of RNA. We show by sedimentation and gel filtration that Nck1 and CE are together in a larger complex, that the complex can assemble <i>in vitro</i> on recombinant Nck1, and Nck1 knockdown disrupts the integrity of the complex. CE and the 5′ kinase are juxtaposed by binding to the adjacent domains of Nck1, and cap homeostasis is inhibited by Nck1 with inactivating mutations in each of these domains. These results identify a new domain of CE that is specific to its function in cytoplasmic capping, and a new role for Nck1 in regulating gene expression through its role as the scaffold for assembly of the cytoplasmic capping complex.</p></div

Model for assembly of the cytoplasmic capping complex.

<p>In this model Nck1 serves as the scaffold for assembly of the cytoplasmic capping complex, with the 5′-kinase and CE juxtaposed by binding to adjacent domains. The ubiquitination site between the second and third SH3 and the SH2 domain for binding tyrosine phosphoproteins (pTyr) are indicated by red arrows. It has yet to be determined how the methyltransferase joins the complex to complete the reaction.</p

Identification of the CE and 5′-kinase binding domains.

<p>(A) The organization of Nck1 (wild type [WT]) is shown together with a series of plasmids expressing HA-tagged forms with inactivating mutations (black box) in each of the functional domains. (B) HEK293 cells were co-transfected with plasmids expressing the indicated forms of Nck1 and bio-cCE. Protein recovered on streptavidin beads was analyzed by Western blotting with antibodies to the Myc tag on bio-cCE and the HA tag on Nck1. (C) Cells were co-transfected with plasmids expressing bio-cCE and HA-tagged wild type Nck1 (WT) or Nck1 with inactivating mutations in the third SH3 domain (M3) or all 3 SH3 domains (3SH3M). Protein recovered on streptavidin beads was analyzed by Western blotting with Alexafluor800-coupled streptavidin (cCE) and anti-HA (Nck1) antibody. Kinase activity was assayed by incubating the recovered proteins with a 23 nt 5′-monophosphate RNA and γ-[³²P]ATP, and capping activity was assayed by incubating recovered proteins with with ATP and α-[³²P]GTP. The products of each reaction were separated on a denaturing polyacrylamide/urea gel and visualized by autoradiography. (D) HEK293 cells were transfected with the plasmids expressing HA-tagged forms of wild-type Nck1, or Nck1 with inactivating mutations in the first (M1) and second (M2) SH3 domains. Complexes recovered on anti-HA beads were analyzed by Western blotting (upper panels), and for 5′-kinase activity as in (C).</p

A functional role for Nck1 in cap homeostasis.

<p>Triplicate cultures of U2OS cells were transfected with plasmids expressing HA-tagged wild-type Nck1, Nck1 mutated in the CE-binding domain (M3), or the 5′-kinase-binding domain (M2). Western blots showing overexpression of each of these proteins are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001933#pbio.1001933.s004" target="_blank">Figure S4</a>. The appearance of uncapped forms of transcripts in the “capping inhibited” pool (grey bars) was determined by their recovery on streptavidin beads after ligation of an RNA adapter and hybridization to a biotinylated antisense DNA oligonucleotide <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001933#pbio.1001933-Mukherjee1" target="_blank">[13]</a>. Each preparation contained an equal amount of uncapped human β-globin mRNA as an internal control and RNA recovered from M3 (A) and M2 (B) expressing cells was analyzed by qRT-PCR for four transcripts that accumulate uncapped forms in cells that are inhibited for cytoplasmic capping (DNAJB1, ILF2, MAPK1, RAB1A). The results are normalized to the signal from cells expressing wild-type Nck1. RNA from M3 (C) or M2 (D) expressing cells was also analyzed by qRT-PCR for three transcripts of the “uninduced” pool whose steady state levels are reduced when cytoplasmic capping is inhibited (TLR1, NME9, S100Z, black bars), one of the transcripts examined in a and b (MAPK1, grey bars), and a control transcript (BOP1, white bars). The results are presented as fold change with respect to wild-type Nck1 and are presented as mean ± standard deviation. The asterisk (*) indicates <i>p</i><0.05 by Student's unpaired two-tailed <i>t</i> test.</p

Identification of Nck1 as a component of the cytoplasmic capping complex.

<p>(A) Cytoplasmic extract from cells expressing bio-cCE was separated on a 10%–50% glycerol gradient. Fractions containing each of these proteins were identified by Western blotting of input fractions with antibodies to the Myc tag on bio-cCE and to Nck1 (upper 2 panels). Streptavidin beads were used to recover bio-cCE from individual fractions and bound proteins were again analyzed by Western blotting with anti-Myc and anti-Nck1 antibodies (lower two panels). (B) Cytoplasmic extract from non-transfected cells was separated on a calibrated Sephacryl S-200 column. Starting with the void volume individual fractions were collected and analyzed by Western blotting with anti-CE and anti-Nck1 antibodies. The missing CE band in fraction 3 was due to sample loss during loading. (C) The fractions indicated with a box at the bottom of (B) were pooled and immunoprecipitated with anti-Nck1 or control IgG. 20% of the immunoprecipitated sample was used for Western blotting with anti-Nck1 antibody and 70% of the immunoprecipitated sample was used for Western blotting with anti-CE antibody. (D) Cytoplasmic extract from non-transfected cells was immunoprecipitated with anti-CE antibody or control IgG, and the recovered proteins were analyzed by Western blotting with anti-Nck1 antibody.</p

Identification of Nck1 binding to cytoplasmic CE.

<p>(A) HEK293 cells were co-transfected with plasmids expressing HA-Nck1 and bio-cCE (lanes 1 and 4), bio-cCEΔ25C (lanes 2 and 5), and bio-cCEΔpro in which the five C-terminal prolines shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001933#pbio-1001933-g001" target="_blank">Figure 1A</a> were deleted (bio-cCEΔpro, lanes 3 and 6). Protein recovered on streptavidin beads was analyzed by Western blotting with anti-Myc (cCE) and anti-HA (Nck1) antibodies (upper two panels). In the lower two panels the bound complexes were assayed as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001933#pbio-1001933-g001" target="_blank">Figure 1</a> for guanylylation and capping activity. The quantified capping activity (mean ± standard deviation, <i>n</i> = 3) for capping assay is shown beneath that autoradiogram. The same results were obtained for each of the modified forms of CE, with <i>p</i>-value<0.05 by Student's <i>t</i> test. (B) Cytoplasmic extract from cells expressing bio-cCE (cCE) and HA-Nck1 was treated ± micrococcal nuclease (MNase) prior to recovery of CE and associated proteins with streptavidin beads. Proteins were analyzed by Western blotting with anti-Myc (cCE) and anti-HA (Nck1) antibodies. (C) HEK293 cells were transfected with plasmids expressing bio-cCE or a protein consisting of two MS2 binding sites fused to the biotinylation sequence present in bio-cCE (MS2-bio) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001933#pbio.1001933-Tsai1" target="_blank">[19]</a>. Proteins recovered on streptavidin beads were analyzed by Western blotting with HRP-streptavidin (bio-cCE and MS2-bio), and with an antibody to endogenous Nck1. (D) The experiment in (C) was repeated except Western blots of recovered proteins were probed with antibodies to Nck2 and Grb2. (E) Cytoplasmic extract was immunoprecipitated with control IgG or anti-Nck1 antibody. 2.5% of input sample and 20% of the immunoprecipitated sample was used for Western blotting to determine Nck1 recovery, and 2.5% of input sample and 70% of the immunoprecipitated sample was assayed by guanylylation to determine CE recovery. The weak guanylylation signal seen in the input sample is due to the presence of a previously described <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001933#pbio.1001933-Otsuka1" target="_blank">[11]</a> inhibitory activity in cytoplasmic extract.</p

Identification of Cytoplasmic Capping Targets Reveals a Role for Cap Homeostasis in Translation and mRNA Stability

The notion that decapping leads irreversibly to messenger RNA (mRNA) decay was contradicted by the identification of capped transcripts missing portions of their 5′ ends and a cytoplasmic complex that can restore the cap on uncapped mRNAs. In this study, we used accumulation of uncapped transcripts in cells inhibited for cytoplasmic capping to identify the targets of this pathway. Inhibition of cytoplasmic capping results in the destabilization of some transcripts and the redistribution of others from polysomes to nontranslating messenger ribonucleoproteins, where they accumulate in an uncapped state. Only a portion of the mRNA transcriptome is affected by cytoplasmic capping, and its targets encode proteins involved in nucleotide binding, RNA and protein localization, and the mitotic cell cycle. The 3′ untranslated regions of recapping targets are enriched for AU-rich elements and microRNA binding sites, both of which function in cap-dependent mRNA silencing. These findings identify a cyclical process of decapping and recapping that we term cap homeostasis