The p53 tumour suppressor is mutated in the majority of human tumours. p53′s proposed role as the guardian of the genome is reflected in its multiple effects on transcription genome stability, cell growth and survival. We show that p53 interacts both physically and functionally with the TFIIH complex. There are multiple protein‐protein contacts, involving two regions of p53 and three subunits of TFIIH, ERCC2 (XPD), ERCC3 (XPB) and p62. p53 and its C‐terminus (amino acids 320–393) inhibit both of the TFIIH helicases and in vitro transcription in the absence of TFIIH. Transcription inhibition is overcome by TFIIH. The N‐terminal region of p53 (1–320), lacking the C‐terminus, is inactive on its own, yet apparently affects the activity of the C‐terminus in the native protein. Interestingly, mutant p53s that are frequently found in tumours are less efficient inhibitors of the helicases and transcription. We hypothesize that the interactions provide an immediate and direct link for p53 to the multiple functions of TFIIH in transcription, DNA repair and possibly the cell cycle.

The Genome Analysis Toolkit (GATK) is a popular set of programs for discovering and genotyping variants from next-generation sequencing data. The current GATK recommendation for RNA sequencing (RNA-seq) is to perform variant calling from individual samples, with the drawback that only variable positions are reported. Versions 3.0 and above of GATK offer the possibility of calling DNA variants on cohorts of samples using the HaplotypeCaller algorithm in Genomic Variant Call Format (GVCF) mode. Using this approach, variants are called individually on each sample, generating one GVCF file per sample that lists genotype likelihoods and their genome annotations. In a second step, variants are called from the GVCF files through a joint genotyping analysis. This strategy is more flexible and reduces computational challenges in comparison to the traditional joint discovery workflow. Using a GVCF workflow for mining SNP in RNA-seq data provides substantial advantages, including reporting homozygous genotypes for the reference allele as well as missing data. Taking advantage of RNA-seq data derived from primary macrophages isolated from 50 cows, the GATK joint genotyping method for calling variants on RNA-seq data was validated by comparing this approach to a so-called “per-sample” method. In addition, pair-wise comparisons of the two methods were performed to evaluate their respective sensitivity, precision and accuracy using DNA genotypes from a companion study including the same 50 cows genotyped using either genotyping-by-sequencing or with the Bovine SNP50 Beadchip (imputed to the Bovine high density). Results indicate that both approaches are very close in their capacity of detecting reference variants and that the joint genotyping method is more sensitive than the per-sample method. Given that the joint genotyping method is more flexible and technically easier, we recommend this approach for variant calling in RNA-seq experiments. Electronic supplementary material The online version of this article (10.1186/s40104-019-0359-0) contains supplementary material, which is available to authorized users.

BackgroundBovine milk fat composition is responsive to dietary manipulation providing an avenue to modify the content of fatty acids and especially some specific unsaturated fatty acid (USFA) isomers of benefit to human health. MicroRNAs (miRNAs) regulate gene expression but their specific roles in bovine mammary gland lipogenesis are unclear. The objective of this study was to determine the expression pattern of miRNAs following mammary gland adaptation to dietary supplementation with 5 % linseed or safflower oil using next generation RNA-sequencing.MethodsTwenty-four Canadian Holstein dairy cows (twelve per treatment) in mid lactation were fed a control diet (total mixed ration of corn:grass silages) for 28 days followed by a treatment period (control diet supplemented with 5 % linseed or safflower oil) of 28 days. Milk samples were collected weekly for fat and individual fatty acid determination. RNA from mammary gland biopsies harvested on day-14 (control period) and on days +7 and +28 (treatment period) from six randomly selected cows per treatment was subjected to small RNA sequencing.ResultsMilk fat percentage decreased significantly (P < 0.001) during treatment with the two diets as compared to the control period. The individual saturated fatty acids C4:0, C6:0, C8:0, C14:0 and C16:0 decreased significantly (P < 0.05) while five USFAs (C14:1, C18:1n11t, C20:3n3, C20:5n3 and CLA:t10c12) increased remarkably (P < 0.05) in response to both treatments. Analysis of 361 million sequence reads generated 321 known bovine miRNAs and 176 novel miRNAs. The expression of fourteen and twenty-two miRNAs was affected (P < 0.05) by linseed and safflower oil treatments, respectively. Seven miRNAs including six up-regulated (bta-miR-199c, miR-199a-3p, miR-98, miR-378, miR-148b and miR-21-5p) and one down-regulated (bta-miR-200a) were found to be regulated (P < 0.05) by both treatments, and thus considered core differentially expressed (DE) miRNAs. The gene targets of core DE miRNAs have functions related to gene expression and general cellular metabolism (P < 0.05) and are enriched in four pathways of lipid metabolism (3-phosphoinositide biosynthesis, 3-phosphoinositide degradation, D-myo-inisitol-5-phosphate metabolism and the superpathway of inositol phosphate compounds).ConclusionOur results suggest that DE miRNAs in this study might be important regulators of bovine mammary lipogenesis and metabolism. The novel miRNAs identified in this study will further enrich the bovine miRNome repertoire and contribute to understanding mammary gland biology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1965-7) contains supplementary material, which is available to authorized users.

Spermatozoa are terminally differentiated cells produced during the complex process of spermatogenesis. Although the role of their residual RNA content is still being debated, this transcriptome may represent a fingerprint of spermatogenesis quality. In the present study, we undertook differential transcript profiling of spermatozoa from fertile bulls with extreme nonreturn rates (NRRs): a low-fertile group, and a high-fertile group. Using the suppression-subtractive hybridization technique in combination with macroarray analysis, we also identified novel genes. Both extreme NRR index groups retained redundant identity, such as ribosomal and mitochondrial sequences, at a statistically significant level. An elevated number of 12S, 18S, and Large Chain R rRNA gene copies were found in low-fertile bulls and validated in spermatozoa by quantitative RT-PCR for a small cohort of bulls with known fertility index. Whereas the high-NRR library exhibited a large proportion (29%) of transcripts associated with known functions (e.g., metabolism, signal transduction, translation, glycosylation, and protein degradation), only 10% of the low-NRR sequences did. This difference is also conveyed by two other categories: 17% Bovine Genome and 48% unknown in the high-NRR library, compared with 3% and 80%, respectively, in the low-NRR library. Some of the unknown transcripts are similar to expressed sequence tags detected in the male reproductive organ of certain plants and retain homology to a putative human protein. Whereas the individual transcriptome profiles may be useful in fertility assessment, these findings also suggest cross-species conservation, could contribute to a better understanding of spermatogenesis, and provide new insights regarding idiopathic infertility.

Regarding bull fertility, establishing an association between in vitro findings and field fertility requires a multi-parametric approach that measures the integrity of various structures and dynamic functions, such as motion characteristics, among others. The heterogeneous RNA pattern of spermatozoa could be used in genomic analysis for evaluating both spermatogenesis and fertility potential of semen, mainly because of the static status of the transcriptome of this particular differentiated cell. In a previous study, we determined that some spermatozoal transcripts identified by PCR-based cDNA subtraction are associated with non-return rate, a field fertility index. In the present study, the microarray technology was used in conjunction with differential RNA transcript extraction. We have shown that among these genes, some transcripts are also associated with the motility status of a population of sperm cells fractionated from the same ejaculate. We highlighted a systematic data analysis and validation scheme important for the identification of significant transcripts in this context. With such an approach, we found that transcripts encoding a serine/threonine testis-specific protein kinase (TSSK6) and a metalloproteinase non coding RNA (ADAM5P ) are associated with high-motility status (P!0.001), also confirmed by quantitative PCR (PZ0.0075). This association was found only when transcripts were extracted using the hot-TRIzol protocol, whereas the cold-TRIzol RNA extract comprised mitochondrial transcripts. These results demonstrate that some transcripts previously identified in association with field fertility are also found associated with in vitro motility provided that a stringent RNA extraction protocol is used.