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      Transcriptome Analysis Reveals the Effect of Long Intergenic Noncoding RNAs on Pig Muscle Growth and Fat Deposition

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          Abstract

          Muscle growth and fat deposition are the two important biological processes in the development of pigs which are closely related to the pig production performance. Long intergenic noncoding RNAs (lincRNAs), with lack of coding potential and the length of at least 200nt, have been extensively studied to play important roles in many biological processes. However, the importance and molecular regulation mechanism of lincRNAs in the process of muscle growth and fat deposition in pigs are still to be further studied comprehensively. In our study, we used the data, including liver, abdominal fat, and longissimus dorsi muscle of 240 days' age of two F2 full-sib female individuals from the white Duroc and Erhualian crossbreed, to identify 581 putative lincRNAs associated with pig muscle growth and fat deposition. The 581 putative lincRNAs shared many common features with other mammalian lincRNAs, such as fewer exons, lower expression levels, and shorter transcript lengths. Cross-tissue comparisons showed that many transcripts were tissue-specific and were involved in the important biological processes in their corresponding tissues. Gene ontology and pathway analysis revealed that many potential target genes (PTGs) of putative lincRNAs were involved in pig muscle growth and fat deposition-related processes, including muscle cell proliferation, lipid metabolism, and fatty acid degradation. In Quantitative Trait Locus (QTLs) analysis, some PTGs were screened from putative lincRNAs, MRPL12 is associated with muscle growth, GCGR and SLC25A10 were associated with fat deposition, and PPP3CA, DPYD, and FGGY were related not only to muscle growth but also to fat deposition. Therefore, it implied that these lincRNAs might participate in the biological processes related to muscle growth or fat deposition through homeostatic regulation of PTGs, but the detailed molecular regulatory mechanisms still needed to be further explored. This study lays the molecular foundation for the in-depth study of the role of lincRNAs in the pig muscle growth and fat deposition and further provides the new molecular markers for understanding the complex biological mechanisms of pig muscle growth and fat deposition.

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          Ab initio reconstruction of transcriptomes of pluripotent and lineage committed cells reveals gene structures of thousands of lincRNAs

          RNA-Seq provides an unbiased way to study a transcriptome, including both coding and non-coding genes. To date, most RNA-Seq studies have critically depended on existing annotations, and thus focused on expression levels and variation in known transcripts. Here, we present Scripture, a method to reconstruct the transcriptome of a mammalian cell using only RNA-Seq reads and the genome sequence. We apply it to mouse embryonic stem cells, neuronal precursor cells, and lung fibroblasts to accurately reconstruct the full-length gene structures for the vast majority of known expressed genes. We identify substantial variation in protein-coding genes, including thousands of novel 5′-start sites, 3′-ends, and internal coding exons. We then determine the gene structures of over a thousand lincRNA and antisense loci. Our results open the way to direct experimental manipulation of thousands of non-coding RNAs, and demonstrate the power of ab initio reconstruction to render a comprehensive picture of mammalian transcriptomes.
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            Evolutionary dynamics and tissue specificity of human long noncoding RNAs in six mammals

            Long intergenic noncoding RNAs (lincRNAs) play diverse regulatory roles in human development and disease, but little is known about their evolutionary history and constraint. Here, we characterize human lincRNA expression patterns in nine tissues across six mammalian species and multiple individuals. Of the 1898 human lincRNAs expressed in these tissues, we find orthologous transcripts for 80% in chimpanzee, 63% in rhesus, 39% in cow, 38% in mouse, and 35% in rat. Mammalian-expressed lincRNAs show remarkably strong conservation of tissue specificity, suggesting that it is selectively maintained. In contrast, abundant splice-site turnover suggests that exact splice sites are not critical. Relative to evolutionarily young lincRNAs, mammalian-expressed lincRNAs show higher primary sequence conservation in their promoters and exons, increased proximity to protein-coding genes enriched for tissue-specific functions, fewer repeat elements, and more frequent single-exon transcripts. Remarkably, we find that ∼20% of human lincRNAs are not expressed beyond chimpanzee and are undetectable even in rhesus. These hominid-specific lincRNAs are more tissue specific, enriched for testis, and faster evolving within the human lineage.
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              Genomic and Transcriptional Co-Localization of Protein-Coding and Long Non-Coding RNA Pairs in the Developing Brain

              Introduction The mammalian genome displays a complex and extensive pattern of interlaced transcription of protein-coding genes and thousands of non-coding RNA (ncRNA; see Materials and Methods for definitions) loci [1]. Exons from ncRNA loci may overlap on the same (sense), or opposite (antisense), strand with exons from other transcripts, including those from protein-coding genes. They may also be contained within introns of other transcripts. Other ncRNAs are transcribed from bidirectional promoters: their transcriptional events, and those for neighbouring transcripts from the opposite strand, are initiated in close genomic proximity. Several recent studies investigated whether cis-antisense, intronic, or bidirectional ncRNAs regulate the transcription of protein-coding genes whose loci they overlap [2],[3]. These report complex relationships between the expression profiles of ncRNAs and their overlapping protein-coding genes in adult mice. Further investigations, however, are clearly needed to investigate other types of ncRNAs, in particular intergenic and long (>200 nt) ncRNAs transcribed from outside protein-coding loci, and those expressed during development. If most long ncRNAs convey biological functions, then what these molecular mechanisms are remain almost completely unknown. For the few with established mechanisms a general theme has emerged of them acting as transcriptional regulators of protein-coding genes (reviewed in [4]). For many such ncRNAs, the genomic location of their transcription has proved key to their mechanism. When promoters of non-coding and coding transcripts are closely juxtaposed on the chromosome, for example, then transcriptional events initiated from them may be coupled. This has been shown to occur following chromatin remodelling of chromosomal domains [5]–[7], or because of collisions between transcriptional machineries processing along sequence in close proximity [8], or because of transcriptional interference when transcription proceeds through a promoter sequence thereby suppressing transcription initiation from it [8]. Other long ncRNAs are cis-regulators of transcription via indirect means involving their participation in ribonucleoprotein complexes [9],[10]. Other long ncRNAs, such as NRON or 7SK, act in trans: they regulate the expression of target genes or gene products from chromosomes other than the ones from which they are transcribed [11]–[13]. Cis-regulation by ncRNAs of protein-coding gene transcription is well-established in imprinting [14] and for developmental genes, such as Dlx5 and Dlx6 [9], yet these represent transcriptional events that overlap on the genome. By way of contrast, we sought statistical evidence that pairs of adjacent, yet distinct, coding and non-coding loci often give rise to separate transcripts with similar spatiotemporal expression patterns indicative of positive co-operativity of transcriptional regulation. (Of course, negative co-operativity by, for example, transcriptional interference is also likely. However, such instances tend to be harder to establish experimentally owing to low levels of ncRNA expression.) We considered that if evidence of transcriptional co-operativity were to be forthcoming then specific pairs of coding and noncoding transcripts could be prioritised for experimentation. In such studies, it is important to demonstrate that long ncRNAs and mRNAs are transcribed exclusively from separate promoters. Otherwise, similarities in their expression profiles may not represent distinct transcriptional events but instead single transcripts spanning both coding and noncoding exons. We recently demonstrated several evolutionary signatures of functionality for a large set of mouse long ncRNAs and their promoters [15]. These long ncRNA sequences are largely full-length [16], map to genomic loci lying outside of protein-coding gene models and consequently are unlikely to act as antisense transcripts of a neighbouring gene locus. Although some of these ncRNAs may result from uncoordinated and inconsequential transcription, evidence of transcriptional regulation [17] and constraints on splicing motifs [15] cannot be explained by such transcriptional ‘noise’. We were interested in whether long intergenic ncRNAs are located randomly with respect to protein-coding genes. If not, this might suggest a trend for long ncRNAs to act in cis with neighbouring protein-coding genes. To improve our chances of detecting non-uniformities of chromosomal location, we considered long ncRNAs whose genomic sequences are evolutionarily constrained and thus are more likely to be functional. If long ncRNAs possess, in general, cis-regulatory roles, one might expect their transcribed genomic regions to lie in proximity to their functionally-linked protein-coding genes, and their tissue expression profiles to be similar. Finally, it might also be expected that functional long ncRNAs would tend to be linked to certain subsets of protein-coding genes that convey particular biological functions. We investigated this cis-regulatory hypothesis for a set of 659 evolutionary constrained long ncRNAs and found large-scale and experimental evidence for co-regulation of non-coding and protein-coding transcript pairs. For the first time, we show that these constrained long ncRNAs are not evenly distributed on the genome but rather tend to be concentrated near to genes with similar expression patterns and from particular functional classes. These findings immediately provide new and unbiased criteria for prioritising long ncRNAs for experimental investigation. Hundreds of constrained long ncRNAs can now be targeted for detailed examination, specifically those that either (i) are expressed in the brain during development and are transcribed in proximity to transcription factor genes, or (ii) are expressed outside of the CNS in adult individuals and that lie adjacent to signalling genes. Results This study examined large numbers of mouse long intergenic ncRNAs, partitioned by the availability or otherwise of evidence for their expression in the brain or during development, and of evidence for sequence constraint. Previous studies had focused specifically on the expression of antisense, bidirectional and intronic ncRNAs in 56 day old adult mice or during mouse embryonic stem cell differentiation [2],[3]. For each set of ncRNA loci we examined the null hypothesis that they are located at random relative to protein-coding genes. Instead, we find strong and significant co-expression and functional biases. We show experimentally that these biases do not derive from single transcriptional events. Constrained ncRNAs are enriched in predicted RNA secondary structures We started by analysing 3,122 long ncRNAs transcribed from intergenic regions (see Materials and Methods) that, when considered together, exhibit evolutionary constraint [15]. Among these ncRNAs, we then identified 659 long ncRNAs that individually show evidence of constraint (hereafter termed constrained long ncRNAs): individually, their mouse-human nucleotide substitution rate is significantly (p 9 tags). (0.03 MB DOCX) Click here for additional data file. Figure S3 Co-expression of further protein-coding/non-coding RNA transcript pairs in the developing (Panels A, B, C) and adult (Panels D, E, F) CNS. Brightfield images of in situ hybridization from adjacent wild-type sections are shown. (A) Expression of the ncRNA AK082989 appeared ubiquitous in an E13.5 embryo, although Zic4, the adjacent protein coding gene, showed a highly specific pattern of expression in the spinal cord and forebrain at the same time-point, as was described previously (Gaston-Massuet et al., 2005). (B) At P12, Meis1 is only expressed above background levels in the developing cerebellar granule cell layer, where the ncRNA AK042766 is also found expressed. (C) Grik2, however, is expressed ubiquitously in the brain, although the adjacent ncRNA AK047467 is only found at low levels in the cerebellar granule cell layer at P12. (D) Both Hip2 and its paired ncRNA, AK045758, are expressed at high levels in the cortex and the hippocampus. (E) Eif2c3 is ubiquitously expressed in the brain, as is the genomically adjacent transcribed ncRNA, AK047638. (F) Adr also shows a ubiquitous expression pattern, although expression of its paired ncRNA, AK162901, is not detected in the adult brain, consistent with the RT-PCR results (Figure S3). In all cases, the sense strand negative control probe failed to show specific staining (data not shown). Gaston-Massuet, C, Henderson DJ, Greene ND, Copp AJ, (2005) Zic4, a zinc-finger transcription factor, is expressed in the developing mouse nervous system. Dev Dyn, 233: 1110-5. (3.43 MB TIF) Click here for additional data file. Figure S4 RT-PCR and 5′ RACE analysis of protein-coding and non-coding transcripts. (A) Total RNA was purified from the tissues and the developmental time-points indicated. RT-PCR was performed using primers spanning from the 3′ UTR of the protein-coding gene to the adjacent ncRNA genomic sequence. Control amplification using the same primer pairs from genomic DNA (gDNA) and a reaction containing no reverse transcriptase (-RT) is also shown. Importantly, RT-PCR of each protein-coding gene and ncRNA was performed from the same tissue. Apart from Add2/AK013768, no evidence for read-through from the 3′ UTR to the ncRNA was observed that would account for the in situ hybridisation results obtained (Figure 5, Figure 6). (B) 5′ RACE products of all 12 ncRNAs analysed in this study (adjacent pc genes are indicated in brackets). Total RNA was purified from the tissue corresponding to the in situ hybridisation data: adult brain (AK018196 - AK162901), P12 cerebellum (AK149041, AK042766 and AK047467) and E13.5 brain (AK082938, AK049627 and AK082969). In these reactions, a nested reverse primer approximately 300 bp from the predicted ncRNA transcription start site and a nested forward primer specific for the cap-ligated RACE anchor primer was used. A reaction containing no reverse transcriptase (-RT) is also shown for each primer pair. RACE reactions containing no TAP enzyme showed no amplification products (data not shown). (1.16 MB TIF) Click here for additional data file. Table S1 Brain-expressed ncRNAs are more likely to be constrained than ncRNAs expressed elsewhere (χ2-test, p = 3×10−3). This observed bias is independent of the lengths of these constrained ncRNAs since the length distributions of brain- and non-brain-expressed ncRNAs are indistinguishable (p = 0.4, Kolmogorov-Smirnov test). Transcripts classified as constrained or non-constrained were divided further into those transcribed in the same (sense) or opposite (antisense) direction relative to the transcriptional orientation of the most proximal protein-coding gene. Cases where a ncRNA is located near to protein-coding genes that are transcribed on both strands have been excluded. An asterisk (*) indicates a significant association with the direction of transcription of the proximal annotated protein-coding gene (see Materials and Methods). Non-constrained, brain-expressed ncRNAs show no directional preference, whereas non-brain-expressed ncRNAs show a small but significant bias in the opposite orientation (54% transcribed in antisense, p = 6×10−3). (0.03 MB XLS) Click here for additional data file. Table S2 Constrained ncRNAs that are expressed in brain or in nonbrain tissues during development show a significant tendency to lie adjacent to proteincoding genes that are highly expressed in specific tissues (p<10−3; EFDR<0.04). Shown is the significant over-representation of ncRNAs in proximity to protein-coding genes that are expressed in these tissues as a result of the observed densities when compared to expected densities on randomly sampled G+C matched sequences; also shown are the lower and upper confidence intervals (CIs) at the 95% level and the standard deviation. (0.02 MB XLS) Click here for additional data file. Table S3 Brain-expressed and constrained ncRNAs show a tendency to be transcribed near to protein-coding genes expressed in brain tissues. Shown are significant (p-value<10−2, EFDR = 0.53) and non-significant (highlighted in grey) enrichments. The observed densities of ncRNAs transcribed in proximity to protein-coding genes expressed in particular tissues have been compared to expected densities from randomly sampled G+C matched sequences (see Materials and Methods). Also shown are lower and upper confidence intervals (CIs) at the 95% level, and standard deviations (StdDev). Terms highlighted in bold correspond to results shown in Figure 4 (p-value<10−3, EFDR = 0.05). (0.02 MB XLS) Click here for additional data file. Table S4 Experimental EST and CAGE TC (tag cluster) support for six non-coding transcripts (AK018196, AK045528, AK013768, AK149041, AK082938, AK049627) for which in situ hybridizations (ISHs) were performed (see Figure 5, Figure 6). Each of the six brain-derived and evolutionarily constrained ncRNA transcripts was further investigated for additional experimental evidence in the form of ESTs and CAGE TCs and the results are summarized in separate tables. For each EST and CAGE TC, its accession code, coordinates, strand, tissue type and stage are reported, and additionally for each EST its position (5′ or 3′) relative to the ncRNA is shown. (0.08 MB XLS) Click here for additional data file. Table S5 ncRNA data sets used in this study: evolutionary and functional properties. The four sets contain ncRNAs that are (i) constrained and derived from brain-associated tissues, (ii) constrained and derived from tissues outside the CNS, (iii) non-constrained and derived from brain-associated tissues and (iv) non-constrained and derived from tissues outside the CNS. Each ncRNA is represented by its (i) accession code, (ii) genome coordinates (assembly mm5), (iii) strand information and (iv) whether it overlaps with: 1. EvoFold predictions of RNA secondary structure (EvoFold), 2. human copy number variants (CNVs), 3. segmental duplications (SDs), 4. PhastCons multispecies conserved elements (MCSs), and 5. indelpurified segments (IPSs). Overlap is indicated by the integer 1, lack of overlap by 0. (0.35 MB XLS) Click here for additional data file. Table S6 ncRNA data sets used in this study: accession codes of all ncRNAs in these four data sets. The four sets contain ncRNAs that are (i) constrained and derived from brain-associated tissues, (ii) constrained and derived from tissues outside the CNS, (iii) non-constrained and derived from brain-associated tissues and (iv) non-constrained and derived from tissues outside the CNS. In particular, the two unconstrained data sets are listed in their entireties since in Table S5 only those that are homologous to human sequence are shown. (0.16 MB XLS) Click here for additional data file. Text S1 Functional associations and transcript read-through. (0.03 MB DOC) Click here for additional data file.
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                Author and article information

                Contributors
                Journal
                Biomed Res Int
                Biomed Res Int
                BMRI
                BioMed Research International
                Hindawi
                2314-6133
                2314-6141
                2019
                25 June 2019
                : 2019
                : 2951427
                Affiliations
                1Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of the Ministry of Education and Key Laboratory of Swine Genetics and Breeding of the Ministry of Agriculture, Huazhong Agricultural University, Wuhan, China
                2The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China
                Author notes

                Academic Editor: Rita Casadio

                Author information
                https://orcid.org/0000-0002-8030-8632
                Article
                10.1155/2019/2951427
                6614983
                31341893
                b4b022a6-67db-45ca-b17f-eaa5e3c54c69
                Copyright © 2019 Guoting Chen et al.

                This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 9 March 2019
                : 1 June 2019
                Funding
                Funded by: National Natural Science Foundation of China
                Award ID: 31872322
                Award ID: 31601859
                Funded by: Fundamental Research Funds for the Central Universities
                Award ID: 2662017PY030
                Funded by: National High Technology Research and Development Plan
                Award ID: 863
                Award ID: 2011AA100302
                Funded by: China Postdoctoral Science Foundation Funded Project
                Award ID: 2016M592344
                Categories
                Research Article

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