Suppression of the rubberband Unc phenotype by <i>mfap-1</i> mutations.

<p>Suppression of the rubberband Unc phenotype by <i>mfap-1</i> mutations.</p

<i>mfap-1(n4564)</i> and <i>mfap-1(n5214)</i> are weak mutations that act together to suppress the rubberband Unc phenotype of <i>unc-93(e1500)</i> animals.

<p>All transgenes were expressed in <i>mfap-1(n4564 n5214); unc-93(e1500)</i> animals. Two lines were obtained and analyzed for transgene <i>P_myo-3 mfap-1 cDNA (n4564 n5214)::GFP</i>.</p

Mutations in <i>mfap-1</i> and <i>uaf-1</i> interact to affect the locomotion of <i>unc-93(e1500)</i> animals.

*<p>Sterile at 20°C.</p>**<p>Inviable.</p

MFAP-1 is a nuclear protein expressed in multiple tissues.

<p>(A) A 2.5 kb 5′ promoter region upstream of the start codon of <i>mfap-1</i> was used to drive the expression of a transcriptional fusion GFP reporter in transgenic animals. The tissues in which the GFP reporter was expressed are labeled. This figure shows a montage of two photographs, which were pseudo-colored from a gray-scale image to a green-scale image using Adobe Photoshop to reflect the level of GFP expression. (B) Transgene experiments were performed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002827#s4" target="_blank">Materials and Methods</a>. The <i>C. elegans</i> MFAP-1::GFP fusion protein, when expressed in body-wall muscles using a <i>myo-3</i> promoter in either wild-type or <i>mfap-1(n4564 n5214)</i> animals of mixed developmental stages, was exclusively localized in nuclei (left and middle panels). Similarly, human MFAP-1::GFP fusion protein, when expressed in the body-wall muscles of <i>mfap-1(n4564 n5214)</i> animals, was also exclusively localized in nuclei (right panels). Lower panels show typical nuclear GFP signals in adult transgenic animals. Genetic backgrounds and transgenes are indicated.</p

Genetic mapping, cloning, and identification of <i>mfap-1</i>.

<p>(A) Genomic location of the <i>n4564 n5214</i> mutation based on genetic mapping using visible markers and SNPs (<i>e.g.</i>, SNP T01H8: 7110 on the left and SNP F36A2: 3334 on the right). Cosmids tested in rescue experiments are labeled. Only cosmid <i>F43G9</i> rescued the three phenotypic characteristics of <i>mfap-(n4564 n5214)</i> individuals: suppression of the Unc phenotype of <i>unc-93(e1500)</i> animals; partial sterility at 20°C; and temperature-sensitive lethality at 25°C. (B) Transgene rescue experiments. Transgenes (<i>nEx</i>) were injected into the gonads of <i>mfap-1(n4564 n5214); unc-93(e1500)</i> animals, and lines stably transmitting the transgenes were established. Transgenic lines were analyzed for the rescue of each of three abnormalities: suppression of the Unc phenotype of <i>unc-93(e1500)</i> animals; partial sterility at 20°C; and temperature-sensitive lethality at 25°C. <i>mut</i>: the <i>n4564 n5214</i> mutation. (C) Gene structure of <i>mfap-1</i>. Black boxes: coding exons. Open boxes: 5′ and 3′ UTRs. Positions of start (ATG) and stop codons (TGA) are indicated. The sites of the <i>n4564 n5214</i> mutation, the <i>n5214</i> mutation and the <i>tm3456</i>Δ deletion are labeled. (D) Sequence alignment of predicted MFAP-1 proteins from <i>C. elegans</i>, <i>Drosophila</i>, chicken and human. *: amino acids mutated in <i>mfap-1(n4564 n5214)</i> mutants. Amino acids conserved in at least three orthologs are darkly shaded, while amino acids with similar physical properties or conserved in two orthologs are lightly shaded.</p

Reducing <i>mfap-1</i> expression by RNA interference alters the splicing of <i>tos-1</i>.

<p>(A) RT-PCR experiments showing the effect of reducing <i>mfap-1</i> expression by RNAi feeding on <i>tos-1</i> alternative splicing. <i>tos-1</i> splice isoforms are illustrated on right. (B) RT-PCR experiments showing the effect of reducing <i>mfap-1</i> expression by RNAi feeding on the recognition of the cryptic 3′ splice site of <i>tos-1</i> intron 1.</p

<i>mfap-1(n4564 n5214)</i> affects the splicing of <i>tos-1</i>.

<p>(A) RT-PCR experiments showing the effect of <i>mfap-1(n4564 n5214)</i> on <i>tos-1</i> alternative splicing. <i>tos-1</i> splice isoforms are illustrated on the right. (B) The molar ratios of all <i>tos-1</i> splice isoforms with intron 1 retention (isoforms 1 and 2), presented as a percentage of all isoforms combined (Isoforms 1, 2, 3, 4 and 5). Error bars: standard deviations. *p<0.05. (C) The molar ratios of all <i>tos-1</i> splice isoforms with exon 3 skipping (isoforms 2 and 5), presented as a percentage of all isoforms combined (isoforms 1, 2, 3, 4 and 5). Error bars: standard deviations. *p<0.05. (D) RT-PCR experiments showing the effect of the <i>mfap-1(n4564 n5214)</i> mutation on the recognition of the cryptic 3′ splice site of <i>tos-1</i> intron 1. For all analyses, isoform intensities were obtained by analyzing biological duplicates or triplicates using NIH ImageJ software.</p

DataSheet1_Two novel variants in CEP152 caused Seckel syndrome 5 in a Chinese family.doc

Background: Seckel syndrome (SCKL) is a rare autosomal recessive inherited disorder, which is mainly characterized by intrauterine and postnatal growth restrictions, microcephaly, intellectual disability, and a typical “bird-head” facial appearance. Here, we aimed to identify the genetic etiology of a family with suspected SCKL.Methods: This study enrolled a Chinese family suspected of SCKL with their detailed family history and clinical data. We performed karyotype analysis, copy number variation sequencing (CNV-seq), and trio whole-exome sequencing (WES) to explore the genetic etiology in the proband. Furthermore, the quantitative real-time polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) were conducted to confirm the pathogenicity of novel variants.Results: The karyotype analysis and CNV-seq were normal in the proband. Two novel variants in CEP152, c.1060C>T (p.Arg354*) and c.1414-14A>G, were identified in the proband through trio-WES. The qPCR results showed that the total CEP152 mRNA expression levels were significantly reduced in c.1060C>T (p.Arg354*) and c.1414-14A>G compared with healthy control individuals. Moreover, aberrant skipping of exon 12 due to the non-canonical splice-site variant was revealed by RT-PCR and Sanger sequencing.Conclusion: Our findings expanded pathogenic variant spectra in SCKL and offered new insights into the pathogenicity of a non-classical splice-site variant in CEP152, which provided additional information for helping the family improve pregnancy plans in the future.</p

The survival motor neuron gene <i>smn-1</i> interacts with the U2AF large subunit gene <i>uaf-1</i> to regulate <i>Caenorhabditis elegans</i> lifespan and motor functions

<div><p>Spinal muscular atrophy (SMA), the most frequent human congenital motor neuron degenerative disease, is caused by loss-of-function mutations in the highly conserved survival motor neuron gene <i>SMN1</i>. Mutations in <i>SMN</i> could affect several molecular processes, among which aberrant pre-mRNA splicing caused by defective snRNP biogenesis is hypothesized as a major cause of SMA. To date little is known about the interactions of <i>SMN</i> with other splicing factor genes and how SMN affects splicing <i>in vivo</i>. The nematode <i>Caenorhabditis elegans</i> carries a single ortholog of <i>SMN</i>, <i>smn-1</i>, and has been used as a model for studying the molecular functions of SMN. We analyzed RNA splicing of reporter genes in an <i>smn-1</i> deletion mutant and found that <i>smn-1</i> is required for efficient splicing at weak 3′ splice sites. Genetic studies indicate that the defective lifespan and motor functions of the <i>smn-1</i> deletion mutants could be significantly improved by mutations of the splicing factor U2AF large subunit gene <i>uaf-1</i>. In <i>smn-1</i> mutants we detected a reduced expression of U1 and U5 snRNAs and an increased expression of U2, U4 and U6 snRNAs. Our study verifies an essential role of <i>smn-1</i> for RNA splicing <i>in vivo</i>, identifies the <i>uaf-1</i> gene as a potential genetic modifier of <i>smn-1</i> mutants, and suggests that SMN-1 has multifaceted effects on the expression of spliceosomal snRNAs.</p></div