Systematic analysis of alternative first exons in plant genomes

Background  

Alternative splicing (AS) contributes significantly to protein diversity, by selectively using different combinations of exons of the same gene under certain circumstances. One particular type of AS is the use of alternative first exons (AFEs), which can have consequences far beyond the fine-tuning of protein functions. For example, AFEs may change the N-termini of proteins and thereby direct them to different cellular compartments. When alternative first exons are distant, they are usually associated with alternative promoters, thereby conferring an extra level of gene expression regulation. However, only few studies have examined the patterns of AFEs, and these analyses were mainly focused on mammalian genomes. Recent studies have shown that AFEs exist in the rice genome, and are regulated in a tissue-specific manner. Our current understanding of AFEs in plants is still limited, including important issues such as their regulation, contribution to protein diversity, and evolutionary conservation.     

TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression

Intestinal inflammation is a vital precipitating factor of colorectal cancer (CRC), but the underlying mechanisms are still elusive. TANK-binding kinase 1 (TBK1) is a core enzyme downstream of several inflammatory signals. Recent studies brought the impacts of TBK1 in malignant disease to the forefront, we found aberrant TBK1 expression in CRC is correlated with CRC progression. TBK1 inhibition impaired CRC cell proliferation, migration, drug resistance and tumor growth. Bioinformatic analysis and experiments in vitro showed overexpressed TBK1 inhibited mTORC1 signaling activation in CRC along with elevated GLUT1 expression without inducing GLUT1 translation. TBK1 mediated mTORC1 inhibition induces intracellular autophagy, which in turn decreasing GLUT1 degradation. As a rescue, blocking of autophagosome and retromer respectively via autophagy-related gene 7 (ATG7) or TBC1 Domain Family Member 5 (TBC1D5) silence diminished the regulation of TBK1 to GLUT1. GLUT1 staining presented that TBK1 facilitated GLUT1 membrane translocation which subsequently enhanced glucose consumption. Inhibitor of TBK1 also decreased GLUT1 expression which potentiated drug-sensitivity of CRC cell. Collectively, TBK1 facilitates glucose consumption for supporting CRC progression via initiating mTORC1 inhibition induced autophagy which decreases GLUT1 degradation and increases GLUT1 membrane location. The adaptive signaling cascade between TBK1 and GLUT1 proposes a new strategy for CRC therapy.     

A de novo germline mutation of APC for inheritable colon cancer in a Chinese family using multigene next generation sequencing

Inheritable colorectal cancers (CRC) accounted for about 20% of the CRC cases, such as hereditary nonpolyposis colorectal cancer (HNPCC), Gardner syndrome and familial adenomatous polyposis (FAP). A four-generation Han Chinese family was found affected with polyposis in colons. Inferred from the pedigree structure, the disease in this family showed an autosomal dominant inheritance model. To locate the causal mutations in this family, genomic DNAs were extracted and the next generation sequencing for 5 genes relating to colon cancer performed by Ion Torrent Personal Genome Machine with a 314 chip. The reads were aligned with human reference genome hg19 to call variants in the 5 genes. After analysis, 14 variants were detected in the sequenced sample and 13 been collected in dbSNP database and assigned with a rs identification number. In these variants, 9 were synonymous, 4 missense and 1 non-sense. In them, 2 rare variants (c.694C>T in APC and c.1690A>G in MSH2) might be the putative causal mutations for familial adenomatous polyposis (FAP) since the rarity of the mutated allele in normal controls. c.694C>T was detected in only affected members and generated a premature stop codon in APC. It should be a de novo germline mutation making APC containing this stop codon as targets for nonsense-mediated mRNA decay (NMD). c.1690A>G in MSH2 was not only detected in affected members, but also in normal ones in the family. Functional prediction revealed that the amino acid affected by this variant had no effect on the function of MSH2. Here, we report a de novo germline mutation of APC as the causal variant in a Chinese family with inheritable colon cancer by the next generation sequencing.     

Identification and characterization of novel NuMA isoforms

The large nuclear mitotic apparatus (NuMA) has been investigated for over 30 years with functions related to the formation and maintenance of mitotic spindle poles during mitosis. However, the existence and functions of NuMA isoforms generated by alternative splicing remains unclear. In the present work, we show that at least seven NuMA isoforms (categorized into long, middle and short groups) generated by alternative splicing from a common NuMA mRNA precursor were discovered in HeLa cells and these isoforms differ mainly at the carboxyl terminus and the coiled-coil domains. Two “hotspot” exons with molecular mass of 3366-nt and 42-nt tend to be spliced during alternative splicing in long and middle groups. Furthermore, full-length coding sequences of long and middle NuMA obtained by using fusion PCR were constructed into GFP-tagged vector to illustrate their cellular localization. Long NuMA mainly localized in the nucleus with absence from nucleoli during interphase and translocated to the spindle poles in mitosis. Middle NuMA displayed the similar cell cycle-dependent distribution pattern as long NuMA. However, expression of NuMA short isoforms revealed a distinct subcellular localization. Short NuMA were present in the cytosol during the whole cycle, without colocalization with mitotic apparatus. These results have allowed us tentatively to explore a new research direction for NuMA’s various functions.     

Exosome-derived small non-coding RNAs reveal immune response upon grafting transplantation in Pinctada fucata (Mollusca)

Exosomes, a subset of small extracellular vesicles, carry various nucleic acids, proteins, lipids, amino acids and metabolites. They function as a mode of intercellular communication and molecular transfer. Exosome cargo molecules, including small non-coding RNAs (sncRNAs), are involved in the immune response in various organisms. However, the role of exosome-derived sncRNAs in immune responses in molluscs remains unclear. Here, we aimed to reveal the sncRNAs involved in the immune response during grafting transplantation by the pearl oyster Pinctada fucata. Exosomes were successfully extracted from the P. fucata haemolymph during graft transplantation. Abundant microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs) were simultaneously discovered in P. fucata exosomes by small RNA sequencing. The expression patterns of the miRNAs and piRNAs at the grafting and initial stages were not substantially different, but varied significantly between the initial and later stages. Target prediction and functional analysis indicate that these miRNAs and piRNAs are related to immune response upon grafting transplantation, whereas piRNAs may also be associated with transposon silencing by targeting with genome transposon elements. This work provides the basis for a functional understanding of exosome-derived sncRNAs and helps to gain further insight into the PIWI/piRNA pathway function outside of germline cells in molluscs.     

Multiple N-glycans cooperate in balancing misfolded BRI1 secretion and ER retention

Key message

N-glycans play a protective or monitoring role according to the folding state of associated protein or the distance from structural defects.

Abstract

Asparagine-linked (Asn/N-) glycosylation is one of the most prevalent and complex protein modifications and the associated N-glycans play crucial roles on protein folding and secretion. The studies have shown that many glycoproteins hold multiple N-glycans, yet little is known about the redundancy of N-glycans on a protein. In this study, we used BRI1 to decipher the roles of N-glycans on protein secretion and function. We found that all 14 potential N-glycosylation sites on BRI1 were occupied with oligosaccharides. The elimination of single N-glycan had no obvious effect on BRI1 secretion or function except N¹⁵⁴-glycan, which resulted in the retention of BRI1 in the endoplasmic reticulum (ER), similar to the loss of multiple highly conserved N-glycans. To misfolded bri1, the absence of N-glycans next to local structural defects enhanced the ER retention and the artificial addition of N-glycan could help the misfolded bri1-GFPs exiting from the ER, indicating that the N-glycans might serve as steric hindrance to protect the structure defects from ER recognition. We also found that the retention of misfolded bri1-9 by lectins and chaperones in the ER relied on the presence of multiple N-glycans distal to the local defects. Our findings revealed that the N-glycans might play a protective or monitoring role according to the folding state of associated protein or the distance from structural defects.

     

GAAD: A Gene and Autoimmiune Disease Association Database

Autoimmune diseases (ADs) arise from an abnormal immune response of the body against substances and tissues normally present in the body. More than a hundred of ADs have been described in the literature so far. Although their etiology remains largely unclear, various types of ADs tend to share more associated genes with other types of ADs than with non-AD types. Here we present GAAD, a gene and AD association database. In GAAD, we collected 44,762 associations between 49 ADs and 4249 genes from public databases and MEDLINE documents. We manually verified the associations to ensure the quality and credibility. We reconstructed and recapitulated the relationships among ADs using their shared genes, which further validated the quality of our data. We also provided a list of significantly co-occurring gene pairs among ADs; with embedded tools, users can query gene co-occurrences and construct customized co-occurrence network with genes of interest. To make GAAD more straightforward to experimental biologists and medical scientists, we extracted additional information describing the associations through text mining, including the putative diagnostic value of the associations, type and position of gene polymorphisms, expression changes of implicated genes, as well as the phenotypical consequences, and grouped the associations accordingly. GAAD is freely available at http://gaad.medgenius.info.