Yufei Zhu

Length of the ORF, position of the first AUG and the Kozak motif are important factors in potential dual-coding transcripts.

A single mammalian transcript normally encodes one protein, but the transcript of GNAS (G-protein α-subunit) contains two reading frames and produces two structurally unrelated proteins, XLαs and ALEX. No other confirmed GNAS-like dual-coding transcripts have been reported to date, even though many such candidate genes have been predicted by bioinformatics analysis. In this study, we constructed a series of vectors to test how two protein products were translated from a single transcript in vitro. The length of the ORF (open reading frame), position of the first AUG and the Kozak motif were found to be important factors. These factors, as well as 55-bp NMD (nonsense-mediated mRNA decay) rule, were used in a bioinformatics search for candidate dual-coding transcripts. A total of 1307, 750 and 474 two-ORF-containing transcripts were found in human, mouse and rat, respectively, of which 170, 89 and 70, respectively, were found to be potential dual-coding transcripts. Most transcripts showed low conservation among species. Interestingly, dual-coding transcripts were significantly enriched for transcripts from the zinc-finger protein family, which are usually DNA-binding proteins involved in regulation of the transcription process.

Nonsense‐mediated decay targets have multiple sequence‐related features that can inhibit translation

Nonsense‐mediated mRNA decay (NMD) is a surveillance system that eliminates transcripts with premature termination codons. In this study, we show that mRNAs targeted by NMD are also suppressed at the translational level. The low translational efficiency (TE) is a consequence of multiple features acting in concert, including low translation initiation rate, mediated by 5′ secondary structure and by use of weak initiation sites, and low translation elongation speed, mediated by low codon usage bias. Despite low elongation rates, NMD transcripts show low ribosome density in the coding sequence, probably owing to low initiation rates, high abortion rates or rapid transit of the ribosome following initiation failure. The low TE is observed in the absence of NMD and is not explained by low transcript abundance. Translational inefficiency is flexible, such that NMD targets have increased TE upon starvation. We propose that the low TE predisposes to NMD and/or that it is part of a mechanism for regulation of NMD transcripts.

COL4A3 mutations cause focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a histologically identifiable glomerular injury often leading to proteinuria and renal failure. To identify its causal genes, whole-exome sequencing and Sanger sequencing were performed on a large Chinese cohort that comprised 40 FSGS families, 50 sporadic FSGS patients, 9 independent autosomal recessive Alports syndrome (ARAS) patients, and 190 ethnically matched healthy controls. Patients with extrarenal manifestations, indicating systemic diseases or other known hereditary renal diseases, were excluded. Heterozygous COL4A3 mutations were identified in five (12.5%) FSGS families and one (2%) sporadic FSGS patient. All identified mutations disrupted highly conserved protein sequences and none of them was found in either public databases or the 190 healthy controls. Of the FSGS patients with heterozygous COL4A3 mutations, segmental thinning of the glomerular base membrane (GBM) was only detected in the patient with electronic microscopy examination results available. Five ARAS patients (55.6%) had homozygous or compound-heterozygous mutations in COL4A3 or COL4A4. Serious changes in the GBM, hearing loss, and ocular abnormalities were found in 100%, 80%, and 40% of the ARAS patients, respectively. Overall, a new subgroup of FSGS patients resulting from heterozygous COL4A3 mutations was identified. The mutations are relatively frequent in families diagnosed with inherited forms of FSGS. Thus, we suggest screening for COL4A3 mutations in familial FSGS patients.

SKAP2, a novel target of HSF4b, associates with NCK2/F-actin at membrane ruffles and regulates actin reorganization in lens cell.

In addition to roles in stress response, heat shock factors (HSFs) play crucial roles in differentiation and development. Heat shock transcription factor 4 (HSF4) deficiency leads to defect in lens epithelial cell (LEC) differentiation and cataract formation. However, the mechanism remains obscure. Here, we identified Src kinase‐associated phosphoprotein 2 (SKAP2) as a downstream target of HSF4b and it was highly expressed at the anterior tip of lens elongating fibre cells in vivo. The HSF4‐deficient lenses showed reduced SKAP2 expression and defects in actin reorganization. The disassembly of stress fibres and formation of cortical actin fibres are critical for the initiation of LEC differentiation. SKAP2 localized at actin‐rich ruffles in human LECs (SRA01/04 cells) and knockdown SKAP2 using RNA interference impaired the disassembly of cellular stress fibres in response to fibroblast growth factor (FGF)‐b. Overexpression of SKAP2, but not the N‐terminal deletion mutant of SKAP2, induced the actin remodelling. We further found that SKAP2 interacted with the SH2 domain of non‐catalytic region of tyrosine kinase adaptor protein 2 (NCK2) via its N‐terminus. The complex of SKAP2‐NCK2‐F‐actin accumulated at the leading edge of the lamellipodium, where FGF receptors and focal adhesion were also recruited. These results revealed an essential role for HSF4‐mediated SKAP2 expression in the regulation of actin reorganization during lens differentiation, likely through a mechanism that SKAP2 anchors the complex of NCK2/focal adhesion to FGF receptors at the lamellipodium in lens epithelial cells.

Alternative splicing at GYNNGY 5′ splice sites: more noise, less regulation

Numerous eukaryotic genes are alternatively spliced. Recently, deep transcriptome sequencing has skyrocketed proportion of alternatively spliced genes; over 95% human multi-exon genes are alternatively spliced. One fundamental question is: are all these alternative splicing (AS) events functional? To look into this issue, we studied the most common form of alternative 5′ splice sites—GYNNGYs (Y = C/T), where both GYs can function as splice sites. Global analyses suggest that splicing noise (due to stochasticity of splicing process) can cause AS at GYNNGYs, evidenced by higher AS frequency in non-coding than in coding regions, in non-conserved than in conserved genes and in lowly expressed than in highly expressed genes. However, ∼20% AS GYNNGYs in humans and ∼3% in mice exhibit tissue-dependent regulation. Consistent with being functional, regulated GYNNGYs are more conserved than unregulated ones. And regulated GYNNGYs have distinctive sequence features which may confer regulation. Particularly, each regulated GYNNGY comprises two splice sites more resembling each other than unregulated GYNNGYs, and has more conserved downstream flanking intron. Intriguingly, most regulated GYNNGYs may tune gene expression through coupling with nonsense-mediated mRNA decay, rather than encode different proteins. In summary, AS at GYNNGY 5′ splice sites is primarily splicing noise, and secondarily a way of regulation.

Evidence for OTUD-6B participation in B lymphocytes cell cycle after cytokine stimulation.

Deubiquitinating enzymes (DUBs) are important regulators of cell proliferation. Here we identified a functional deubiquitinating enzyme, ovarian tumor domain-containing 6B (OTUD-6B). Mutation of the conserved Cys residue abolished its deubiquitinating activity in vitro. Otud-6b expression was induced with cytokine stimulation in both mouse Ba/F3 cells and primary B lymphocytes followed a rapid decrease. This rapid decrease was partially facilitated by tristetraprolin (TTP) destabilization of Otud-6b mRNA through AU-rich motifs. Enforced expression of OTUD-6B in Ba/F3 cells could block cell proliferation by arresting cells in G1 phase. In addition, cyclin D2 level was down-regulated when OTUD-6B WT was overexpressed. Therefore, down-regulation of Otud-6b expression after prolonged cytokine stimulation may be required for cell proliferation in B lymphocytes.

Genome-Wide Association Studies of HIV-1 Host Control in Ethnically Diverse Chinese Populations

Genome-wide association studies (GWASs) have revealed several genetic loci associated with HIV-1 outcome following infection (e.g., HLA-C at 6p21.33) in multi-ethnic populations with genetic heterogeneity and racial/ethnic differences among Caucasians, African-Americans, and Hispanics. To systematically investigate the inherited predisposition to modulate HIV-1 infection in Chinese populations, we performed GWASs in three ethnically diverse HIV-infected patients groups (i.e., HAN, YUN, and XIN, N = 538). The reported loci at 6p21.33 was validated in HAN (e.g., rs9264942, P = 0.0018). An independent association signal (rs2442719, P = 7.85 × 10−7, HAN group) in the same region was observed. Imputation results suggest that haplotype HLA-B*13:02/C*06:02, which can partially account for the GWAS signal, is associated with lower viral load in Han Chinese. Moreover, several novel loci were identified using GWAS approach including the top association signals at 6q13 (KCNQ5, rs947612, P = 2.15 × 10−6), 6p24.1 (PHACTR1, rs202072, P = 3.8 × 10−6), and 11q12.3 (SCGB1D4, rs11231017, P = 7.39 × 10−7) in HAN, YUN, and XIN groups, respectively. Our findings imply shared or specific mechanisms for host control of HIV-1 in ethnically diverse Chinese populations, which may shed new light on individualized HIV/AIDS therapy in China.

Screening of Kozak-motif-located SNPs and analysis of their association with human diseases

The Kozak motif, which is located near the translational start codon, often regulates the protein translation. Moreover, it is believed that the conserved positions -3 and +4 contribute the most. Since changes that occur in this motif have a great impact on protein yield and in some cases are associated with disease, we screened the human SNP database for all Kozak-motif-located SNPs (kSNPs) and focused on the strong-changed kSNPs (sckSNPs). Many intron-located and synonymous SNPs are reported to be associated with disease, though the mechanisms underlying these associations are poorly understood. Here, we performed haplotype analysis on sckSNP-containing genes and found that there are some sckSNPs that exist in the same haplotype blocks of reported intron-located and synonymous disease-associated SNPs, indicating that those kSNPs could be a true risk factor for disease-association by affecting the efficiency of protein expression. Our findings provide a candidate explanation for how diseases are associated with intron-located and synonymous SNPs.

Uncovering the Rare Variants of DLC1 Isoform 1 and Their Functional Effects in a Chinese Sporadic Congenital Heart Disease Cohort

Congenital heart disease (CHD) is the most common birth defect affecting the structure and function of fetal hearts. Despite decades of extensive studies, the genetic mechanism of sporadic CHD remains obscure. Deleted in liver cancer 1 (DLC1) gene, encoding a GTPase-activating protein, is highly expressed in heart and essential for heart development according to the knowledge of Dlc1-deficient mice. To determine whether DLC1 is a susceptibility gene for sporadic CHD, we sequenced the coding region of DLC1 isoform 1 in 151 sporadic CHD patients and identified 13 non-synonymous rare variants (including 6 private variants) in the case cohort. Importantly, these rare variants (8/13) were enriched in the N-terminal region of the DLC1 isoform 1 protein. Seven of eight amino acids at the N-terminal variant positions were conserved among the primates. Among the 9 rare variants that were predicted as “damaging”, five were located at the N-terminal region. Ensuing in vitro functional assays showed that three private variants (Met360Lys, Glu418Lys and Asp554Val) impaired the ability of DLC1 to inhibit cell migration or altered the subcellular location of the protein compared to wild-type DLC1 isoform 1. These data suggest that DLC1 might act as a CHD-associated gene in addition to its role as a tumor suppressor in cancer.

Combined Analysis with Copy Number Variation Identifies Risk Loci in Lung Cancer

Background. Lung cancer is the most important cause of cancer mortality worldwide, but the underlying mechanisms of this disease are not fully understood. Copy number variations (CNVs) are promising genetic variations to study because of their potential effects on cancer. Methodology/Principal Findings. Here we conducted a pilot study in which we systematically analyzed the association of CNVs in two lung cancer datasets: the Environment And Genetics in Lung cancer Etiology (EAGLE) and the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial datasets. We used a preestablished association method to test the datasets separately and conducted a combined analysis to test the association accordance between the two datasets. Finally, we identified 167 risk SNP loci and 22 CNVs associated with lung cancer and linked them with recombination hotspots. Functional annotation and biological relevance analyses implied that some of our predicted risk loci were supported by other studies and might be potential candidate loci for lung cancer studies. Conclusions/Significance. Our results further emphasized the importance of copy number variations in cancer and might be a valuable complement to current genome-wide association studies on cancer.