Crosstalk among lncRNAs, microRNAs and mRNAs in the muscle ‘degradome’ of rainbow trout

In fish, protein-coding and noncoding genes involved in muscle atrophy are not fully characterized. In this study, we characterized coding and noncoding genes involved in gonadogenesis-associated muscle atrophy, and investigated the potential functional interplay between these genes. Using RNA- Seq, we compared expression pattern of mRNAs, long noncoding RNAs (lncRNAs) and microRNAs of atrophying skeletal muscle from gravid females and control skeletal muscle from age-matched sterile individuals. A total of 852 mRNAs, 1,160 lncRNAs and 28 microRNAs were differentially expressed (DE) between the two groups. Muscle atrophy appears to be mediated by many genes encoding ubiquitin- proteasome system, autophagy related proteases, lysosomal proteases and transcription factors. Transcripts encoding atrogin-1 and mir-29 showed exceptional high expression in atrophying muscle, suggesting an important role in bulk muscle proteolysis. DE genes were co-localized in the genome with strong expression correlation, and they exhibited extensive ‘lncRNA-mRNA’, ‘lncRNA-microRNA’, ‘mRNA-microRNA’ and ‘lncRNA-protein’ physical interactions. DE genes exhibiting potential functional interactions comprised the highly correlated ‘lncRNA-mRNA-microRNA’ gene network described as ‘degradome’. This study pinpoints extensive coding and noncoding RNA interactions during muscle atrophy in fish, and provides valuable resources for future mechanistic studies

Whole-body transcriptome of selectively bred, resistant-, control-, and susceptible-line rainbow trout following experimental challenge with Flavobacterium psychrophilum

Genetic improvement for enhanced disease resistance in fish is an increasingly utilized approach to mitigate endemic infectious disease in aquaculture. In domesticated salmonid populations, large phenotypic variation in disease resistance has been identified but the genetic basis for altered responsiveness remains unclear. We previously reported three generations of selection and phenotypic validation of a bacterial cold water disease (BCWD) resistant line of rainbow trout, designated ARS-Fp-R. This line has higher survival after infection by either standardized laboratory challenge or natural challenge as compared to two reference lines, designated ARS-Fp-C (control) and ARS-Fp-S (susceptible). In this study, we utilized 1.1 g fry from the three genetic lines and performed RNA-seq to measure transcript abundance from the whole body of naive and Flavobacterium psychrophilum infected fish at day 1 (early time-point) and at day 5 post-challenge (onset of mortality). Sequences from 24 libraries were mapped onto the rainbow trout genome reference transcriptome of 46,585 predicted protein coding mRNAs that included 2633 putative immune-relevant gene transcripts. A total of 1884 genes (4.0% genome) exhibited differential transcript abundance between infected and mock-challenged fish (FDR \u3c 0.05) that included chemokines, complement components, tnf receptor superfamily members, interleukins, nod-like receptor family members, and genes involved in metabolism and wound healing. The largest number of differentially expressed genes occurred on day 5 post-infection between naive and challenged ARS-Fp-S line fish correlating with high bacterial load. After excluding the effect of infection, we identified 21 differentially expressed genes between the three genetic lines. In summary, these data indicate global transcriptome differences between genetic lines of naive animals as well as differentially regulated transcriptional responses to infection

Genome-Wide Discovery of Long Non-Coding RNAs in Rainbow Trout.

The ENCODE project revealed that ~70% of the human genome is transcribed. While only 1-2% of the RNAs encode for proteins, the rest are non-coding RNAs. Long non-coding RNAs (lncRNAs) form a diverse class of non-coding RNAs that are longer than 200 nt. Emerging evidence indicates that lncRNAs play critical roles in various cellular processes including regulation of gene expression. LncRNAs show low levels of gene expression and sequence conservation, which make their computational identification in genomes difficult. In this study, more than two billion Illumina sequence reads were mapped to the genome reference using the TopHat and Cufflinks software. Transcripts shorter than 200 nt, with more than 83-100 amino acids ORF, or with significant homologies to the NCBI nr-protein database were removed. In addition, a computational pipeline was used to filter the remaining transcripts based on a protein-coding-score test. Depending on the filtering stringency conditions, between 31,195 and 54,503 lncRNAs were identified, with only 421 matching known lncRNAs in other species. A digital gene expression atlas revealed 2,935 tissue-specific and 3,269 ubiquitously-expressed lncRNAs. This study annotates the lncRNA rainbow trout genome and provides a valuable resource for functional genomics research in salmonids

Number of lncRNA predicted in at least 2 of the 4 datasets and final numbers after merging and removal of redundant sequences.

<p>Number of lncRNA predicted in at least 2 of the 4 datasets and final numbers after merging and removal of redundant sequences.</p

Distribution of sequence length of LncRNAs compared to protein-coding transcripts in rainbow trout.

<p>LncRNAs were shorter than protein-coding genes with (0.821 kb) and (1.636 kb) average length in each, respectively (Left). Distribution of number of exons in LncRNAs compared to that of protein-coding genes. Most LncRNA transcripts (~90%) have only one exon whereas majority of the protein-coding transcripts tend to have two or more exons (Right).</p

Number of exons and average length of lncRNAs in different data sets.

<p>Number of exons and average length of lncRNAs in different data sets.</p

Bioinformatics pipeline used in prediction of rainbow trout lncRNAs.

<p>LncRNAs were predicted from four different transcriptomic datasets, then all putative lncRNAs from all data were blasted against each other. A total of 54,503 non-redundant lncRNAs identified in at least 2 of the 4 data sets were chosen for further analyses in order to increase the confidence of lncRNA prediction. Vertical arrows are pointing toward the subsequent prediction and filtration steps of the workflow. First horizontal arrow pointing toward the right is referring to the number of initial transcripts predicted from the four datasets. Middle six horizontal arrows indicate the number of transcripts filtered at each step and the final horizontal arrow points to the number of putative lncRNAs with significant hits to noncoding-RNA databases from each dataset.</p

RPKM comparison of protein-coding genes and lncRNAs.

<p>Transcript abundance of lncRNAs is lower than that of protein-coding genes. Average RPKM of the most abundant 40,000 genes is 15.69 and 3.49 for protein coding genes and LncRNAs, respectively (Left). Number of tissue-specific lncRNAs and protein-coding genes in various tissues. Expression of lncRNAs and protein-coding genes showed similar patterns among different tissues (Right).</p

Classification of lncRNAs based on their intersection with protein-coding genes and number of lncRNAs in each class.

<p>Diagram on the top is a visual illustration of each class of lncRNAs relative to nearest protein-coding gene(s) based on genomic position and direction of transcripts. Bottom Fig in tabular format presents number of different classes of lncRNAs from each class. Numbers inside brackets following data source references indicate total number of that particular class of lncRNAs. Letters C, D, S, AS and U indicate number of convergent, divergent, sense, anti-sense and transcripts with unknown directionality, respectively.</p

RNA-SEQ Reveals Microrna Expression Signature And Genetic Polymorphism Associated With Growth And Muscle Quality Traits In Rainbow Trout

The role of microRNA expression and genetic variation in microRNA-binding sites of target genes on growth and muscle quality traits is poorly characterized. We used RNA-Seq approach to investigate their importance on 5 growth and muscle quality traits: whole body weight (WBW), muscle yield, muscle crude-fat content, muscle shear force and whiteness. Phenotypic data were collected from 471 fish, representing 98 families (~5 fish/family) from a growth-selected line. Muscle microRNAs and mRNAs were sequenced from 22 families showing divergent phenotypes. Ninety microRNAs showed differential expression between families with divergent phenotypes, and their expression was strongly associated with variation in phenotypes. A total of 204 single nucleotide polymorphisms (SNPs) present in 3\u27 UTR of target genes either destroyed or created novel illegitimate microRNA target sites; of them, 78 SNPs explained significant variation in the aforementioned 5 muscle traits. Majority of the phenotype-associated SNPs were present in microRNA-binding sites of genes involved in energy metabolism and muscle structure. These findings suggest that variation in microRNA expression and/or sequence variation in microRNA binding sites in target genes play an important role in mediating differences in fish growth and muscle quality phenotypes