Normalized long read RNA sequencing in chicken reveals transcriptome complexity similar to human

Background: Despite the significance of chicken as a model organism, our understanding of the chicken transcriptome is limited compared to human. This issue is common to all non-human vertebrate annotations due to the difficulty in transcript identification from short read RNAseq data. While previous studies have used single molecule long read sequencing for transcript discovery, they did not perform RNA normalization and 5'-cap selection which may have resulted in lower transcriptome coverage and truncated transcript sequences. Results: We sequenced normalised chicken brain and embryo RNA libraries with Pacific Bioscience Iso-Seq. 5' cap selection was performed on the embryo library to provide methodological comparison. From these Iso-Seq sequencing projects, we have identified 60 k transcripts and 29 k genes within the chicken transcriptome. Of these, more than 20 k are novel lncRNA transcripts with ~3 k classified as sense exonic overlapping lncRNA, which is a class that is underrepresented in many vertebrate annotations. The relative proportion of alternative transcription events revealed striking similarities between the chicken and human transcriptomes while also providing explanations for previously observed genomic differences. Conclusions: Our results indicate that the chicken transcriptome is similar in complexity compared to human, and provide insights into other vertebrate biology. Our methodology demonstrates the potential of Iso-Seq sequencing to rapidly expand our knowledge of transcriptomics

The 3′ processing factor CstF functions in the DNA repair response

Following DNA damage, mRNA levels decrease, reflecting a coordinated interaction of the DNA repair, transcription and RNA processing machineries. In this study, we provide evidence that transcription and polyadenylation of mRNA precursors are both affected in vivo by UV treatment. We next show that the polyadenylation factor CstF, plays a direct role in the DNA damage response. Cells with reduced levels of CstF display decreased viability following UV treatment, reduced ability to ubiquitinate RNA polymerase II (RNAP II), and defects in repair of DNA damage. Furthermore, we show that CstF, RNAP II and BARD1 are all found at sites of repaired DNA. Our results indicate that CstF plays an active role in the response to DNA damage, providing a link between transcription-coupled RNA processing and DNA repair

Thriving under Stress: Selective Translation of HIV-1 Structural Protein mRNA during Vpr-Mediated Impairment of eIF4E Translation Activity

Translation is a regulated process and is pivotal to proper cell growth and homeostasis. All retroviruses rely on the host translational machinery for viral protein synthesis and thus may be susceptible to its perturbation in response to stress, co-infection, and/or cell cycle arrest. HIV-1 infection arrests the cell cycle in the G2/M phase, potentially disrupting the regulation of host cell translation. In this study, we present evidence that HIV-1 infection downregulates translation in lymphocytes, attributable to the cell cycle arrest induced by the HIV-1 accessory protein Vpr. The molecular basis of the translation suppression is reduced accumulation of the active form of the translation initiation factor 4E (eIF4E). However, synthesis of viral structural proteins is sustained despite the general suppression of protein production. HIV-1 mRNA translation is sustained due to the distinct composition of the HIV-1 ribonucleoprotein complexes. RNA-coimmunoprecipitation assays determined that the HIV-1 unspliced and singly spliced transcripts are predominantly associated with nuclear cap binding protein 80 (CBP80) in contrast to completely-spliced viral and cellular mRNAs that are associated with eIF4E. The active translation of the nuclear cap binding complex (CBC)-bound viral mRNAs is demonstrated by ribosomal RNA profile analyses. Thus, our findings have uncovered that the maintenance of CBC association is a novel mechanism used by HIV-1 to bypass downregulation of eIF4E activity and sustain viral protein synthesis. We speculate that a subset of CBP80-bound cellular mRNAs contribute to recovery from significant cellular stress, including human retrovirus infection

A Mild Form of SLC29A3 Disorder: A Frameshift Deletion Leads to the Paradoxical Translation of an Otherwise Noncoding mRNA Splice Variant

We investigated two siblings with granulomatous histiocytosis prominent in the nasal area, mimicking rhinoscleroma and Rosai-Dorfman syndrome. Genome-wide linkage analysis and whole-exome sequencing identified a homozygous frameshift deletion in SLC29A3, which encodes human equilibrative nucleoside transporter-3 (hENT3). Germline mutations in SLC29A3 have been reported in rare patients with a wide range of overlapping clinical features and inherited disorders including H syndrome, pigmented hypertrichosis with insulin-dependent diabetes, and Faisalabad histiocytosis. With the exception of insulin-dependent diabetes and mild finger and toe contractures in one sibling, the two patients with nasal granulomatous histiocytosis studied here displayed none of the many SLC29A3-associated phenotypes. This mild clinical phenotype probably results from a remarkable genetic mechanism. The SLC29A3 frameshift deletion prevents the expression of the normally coding transcripts. It instead leads to the translation, expression, and function of an otherwise noncoding, out-of-frame mRNA splice variant lacking exon 3 that is eliminated by nonsense-mediated mRNA decay (NMD) in healthy individuals. The mutated isoform differs from the wild-type hENT3 by the modification of 20 residues in exon 2 and the removal of another 28 amino acids in exon 3, which include the second transmembrane domain. As a result, this new isoform displays some functional activity. This mechanism probably accounts for the narrow and mild clinical phenotype of the patients. This study highlights the ‘rescue’ role played by a normally noncoding mRNA splice variant of SLC29A3, uncovering a new mechanism by which frameshift mutations can be hypomorphic

Adaptive Evolution of the Lactose Utilization Network in Experimentally Evolved Populations of Escherichia coli

Adaptation to novel environments is often associated with changes in gene regulation. Nevertheless, few studies have been able both to identify the genetic basis of changes in regulation and to demonstrate why these changes are beneficial. To this end, we have focused on understanding both how and why the lactose utilization network has evolved in replicate populations of Escherichia coli. We found that lac operon regulation became strikingly variable, including changes in the mode of environmental response (bimodal, graded, and constitutive), sensitivity to inducer concentration, and maximum expression level. In addition, some classes of regulatory change were enriched in specific selective environments. Sequencing of evolved clones, combined with reconstruction of individual mutations in the ancestral background, identified mutations within the lac operon that recapitulate many of the evolved regulatory changes. These mutations conferred fitness benefits in environments containing lactose, indicating that the regulatory changes are adaptive. The same mutations conferred different fitness effects when present in an evolved clone, indicating that interactions between the lac operon and other evolved mutations also contribute to fitness. Similarly, changes in lac regulation not explained by lac operon mutations also point to important interactions with other evolved mutations. Together these results underline how dynamic regulatory interactions can be, in this case evolving through mutations both within and external to the canonical lactose utilization network

Allelic Gene Structure Variations in Anopheles gambiae Mosquitoes

Allelic gene structure variations and alternative splicing are responsible for transcript structure variations. More than 75% of human genes have structural isoforms of transcripts, but to date few studies have been conducted to verify the alternative splicing systematically.The present study used expressed sequence tags (ESTs) and EST tagged SNP patterns to examine the transcript structure variations resulting from allelic gene structure variations in the major human malaria vector, Anopheles gambiae. About 80% of 236,004 available A. gambiae ESTs were successfully aligned to A. gambiae reference genomes. More than 2,340 transcript structure variation events were detected. Because the current A. gambiae annotation is incomplete, we re-annotated the A. gambiae genome with an A. gambiae-specific gene model so that the effect of variations on gene coding could be better evaluated. A total of 15,962 genes were predicted. Among them, 3,873 were novel genes and 12,089 were previously identified genes. The gene completion rate improved from 60% to 84%. Based on EST support, 82.5% of gene structures were predicted correctly. In light of the new annotation, we found that approximately 78% of transcript structure variations were located within the coding sequence (CDS) regions, and >65% of variations in the CDS regions have the same open-reading-frame. The association between transcript structure isoforms and SNPs indicated that more than 28% of transcript structure variation events were contributed by different gene alleles in A. gambiae.We successfully expanded the A. gambiae genome annotation. We predicted and analyzed transcript structure variations in A. gambiae and found that allelic gene structure variation plays a major role in transcript diversity in this important human malaria vector

Alternative Splicing at a NAGNAG Acceptor Site as a Novel Phenotype Modifier

Approximately 30% of alleles causing genetic disorders generate premature termination codons (PTCs), which are usually associated with severe phenotypes. However, bypassing the deleterious stop codon can lead to a mild disease outcome. Splicing at NAGNAG tandem splice sites has been reported to result in insertion or deletion (indel) of three nucleotides. We identified such a mechanism as the origin of the mild to asymptomatic phenotype observed in cystic fibrosis patients homozygous for the E831X mutation (2623G>T) in the CFTR gene. Analyses performed on nasal epithelial cell mRNA detected three distinct isoforms, a considerably more complex situation than expected for a single nucleotide substitution. Structure-function studies and in silico analyses provided the first experimental evidence of an indel of a stop codon by alternative splicing at a NAGNAG acceptor site. In addition to contributing to proteome plasticity, alternative splicing at a NAGNAG tandem site can thus remove a disease-causing UAG stop codon. This molecular study reveals a naturally occurring mechanism where the effect of either modifier genes or epigenetic factors could be suspected. This finding is of importance for genetic counseling as well as for deciding appropriate therapeutic strategies

Absence of spermatozoal CD46 protein expression and associated rapid acrosome reaction rate in striped field mice (Apodemus agrarius)

<p>Abstract</p> <p>Background</p> <p>In rodents, the cell surface complement regulatory protein CD46 is expressed solely on the spermatozoal acrosome membrane. Ablation of the CD46 gene is associated with a faster acrosome reaction. Sperm from Apodemus flavicollis (yellow-necked field mice), A. microps (pygmy field mice) and A. sylvaticus (European wood mice) fail to express CD46 protein and exhibit a more rapid acrosome reaction rate than Mus (house mice) or BALB/c mice. A. agrarius (striped field mice) belong to a different Apodemus subgenus and have pronounced promiscuity and large relative testis size. The aim of this study was to determine whether A. agrarius sperm fail to express CD46 protein and, if so, whether A. agrarius have a faster acrosome reaction than Mus.</p> <p>Methods</p> <p>Reverse transcription polymerase chain reaction (RT-PCR) was used to assess whether A. agrarius transcribe testicular CD46 mRNA. RT-PCR was supplemented with 3'- and 5'-rapid amplification of cDNA ends to determine the complete nucleotide sequence of A. agrarius CD46. Fluorescence microscopy was used to assess whether CD46 protein is expressed by A. agrarius sperm. The acrosome status of A. agrarius sperm was calculated over time by immunocytochemistry using peanut agglutinin lectin.</p> <p>Results</p> <p>We demonstrate that A. agrarius mice transcribe two unique alternatively spliced testicular CD46 mRNA transcripts, both lacking exon 7, which differ from those described previously in other Apodemus species. The larger A. agrarius CD46 transcript has an insert between exons 10 and 11 which, if translated, would result in a novel cytoplasmic tail. In addition, A. agrarius CD46 transcripts have an extended AU-rich 3'-untranslated region (UTR) and a truncated 5'-UTR, resulting in failure to express spermatozoal CD46 protein. We show that A. agrarius has a significantly faster spontaneous acrosome reaction rate than A. sylvaticus and Mus.</p> <p>Conclusion</p> <p>Absence of CD46 protein expression is associated with acrosomal instability in rodents. A. agrarius mice express novel CD46 transcripts, resulting in the trade of spermatozoal CD46 protein expression for a rapid acrosome reaction rate, in common with other species of field mice. This provides a strategy to increase competitive sperm advantage for individuals, leading to faster fertilisation in this highly promiscuous genus.</p

High proportion of recurrent germline mutations in the BRCA1 gene in breast and ovarian cancer patients from the Prague area

BACKGROUND: Germline mutations in the BRCA1 and BRCA2 genes have been shown to account for the majority of hereditary breast and ovarian cancers. The purpose of our study was to estimate the incidence and spectrum of pathogenic mutations in BRCA1/2 genes in high-risk Czech families. METHODS: A total of 96 Czech families with recurrent breast and/or ovarian cancer and 55 patients considered to be at high-risk but with no reported family history of cancer were screened for mutations in the BRCA1/2 genes. The entire coding sequence of each gene was analyzed using a combination of the protein truncation test and direct DNA sequencing. RESULTS: A total of 35 mutations in the BRCA1/2 genes were identified in high-risk families (36.5%). Pathogenic mutations were found in 23.3% of breast cancer families and in 59.4% of families with the occurrence of both breast and ovarian cancer. In addition, four mutations were detected in 31 (12.9%) women with early onset breast cancer. One mutation was detected in seven (14.3%) patients affected with both a primary breast and ovarian cancer and another in three (33.3%) patients with a bilateral breast cancer. A total of 3 mutations in BRCA1 were identified among 14 (21.4%) women with a medullary breast carcinoma. Of 151 analyzed individuals, 35 (23.2%) carried a BRCA1 mutation and 9 (6.0%) a BRCA2 mutation. One novel truncating mutation was found in BRCA1 (c.1747A>T) and two in BRCA2 (c.3939delC and c.5763dupT). The 35 identified BRCA1 mutations comprised 13 different alterations. Three recurrent mutations accounted for 71.4% of unrelated individuals with detected gene alterations. The BRCA1 c.5266dupC (5382insC) was detected in 51.4% of mutation positive women. The mutations c.3700_3704del5 and c.181T>G (300T>G) contributed to 11.4% and 8.6% of pathogenic mutations, respectively. A total of eight different mutations were identified in BRCA2. The novel c.5763dupT mutation, which appeared in two unrelated families, was the only recurrent alteration of the BRCA2 gene identified in this study. CONCLUSION: Mutational analysis of BRCA1/2 genes in 151 high-risk patients characterized the spectrum of gene alterations and demonstrated the dominant role of the BRCA1 c.5266dupC allele in hereditary breast and ovarian cancer

A Yeast Model of FUS/TLS-Dependent Cytotoxicity

FUS/TLS is a nucleic acid binding protein that, when mutated, can cause a subset of familial amyotrophic lateral sclerosis (fALS). Although FUS/TLS is normally located predominantly in the nucleus, the pathogenic mutant forms of FUS/TLS traffic to, and form inclusions in, the cytoplasm of affected spinal motor neurons or glia. Here we report a yeast model of human FUS/TLS expression that recapitulates multiple salient features of the pathology of the disease-causing mutant proteins, including nuclear to cytoplasmic translocation, inclusion formation, and cytotoxicity. Protein domain analysis indicates that the carboxyl-terminus of FUS/TLS, where most of the ALS-associated mutations are clustered, is required but not sufficient for the toxicity of the protein. A genome-wide genetic screen using a yeast over-expression library identified five yeast DNA/RNA binding proteins, encoded by the yeast genes ECM32, NAM8, SBP1, SKO1, and VHR1, that rescue the toxicity of human FUS/TLS without changing its expression level, cytoplasmic translocation, or inclusion formation. Furthermore, hUPF1, a human homologue of ECM32, also rescues the toxicity of FUS/TLS in this model, validating the yeast model and implicating a possible insufficiency in RNA processing or the RNA quality control machinery in the mechanism of FUS/TLS mediated toxicity. Examination of the effect of FUS/TLS expression on the decay of selected mRNAs in yeast indicates that the nonsense-mediated decay pathway is probably not the major determinant of either toxicity or suppression.Fidelity Biosciences (Firm)Fidelity Biosciences (Firm) (Research Inititative)ALS Therapy AllianceNational Institutes of Health (U.S.) (NIH 1RC1NS06839)National Institutes of Health (U.S.) (NIH U01NS05225-03)National Institutes of Health (U.S.) (NIH R01NS050557-05)National Institutes of Health (U.S.) (NIH 1RC2NS070342-01)Pierre L. de Bourgknecht ALS Research FoundationNational Science Foundation (U.S.) (NS614192