Small genetic variation affecting mRNA isoforms associated with marbling and meat color in beef cattle.

The aim of this study was to identify mRNA isoforms and small genetic variants that may be affecting marbling and beef color in Nellore cattle. Longissimus thoracis muscle samples from 20 bulls with different phenotypes (out of 80 bulls set) for marbling (moderate (n = 10) and low (n = 10) groups) and beef color (desirable (n = 10) and undesirable (n = 9) group) traits were used to perform transcriptomic analysis using RNA sequencing. Fourteen and 15 mRNA isoforms were detected as differentially expressed (DE) (P-value ≤ 0.001) between divergent groups for marbling and meat color traits, respectively. Some of those DE mRNA isoforms have shown sites of splicing modified by small structural variants as single nucleotide variant (SNV), insertion, and/or deletion. Enrichment analysis identified metabolic pathways, such as O2/CO2 exchange in erythrocytes, tyrosine biosynthesis, and phenylalanine degradation. The results obtained suggest potential key regulatory genes associated with these economically important traits for the beef industry and for the consumer.

Expression of myosin heavy chain isoform mRNA transcripts in the masseter and medial pterygoid muscles / Expresión de transcripciones de ARNm de isoforma de cadena pesada de miosina en los músculos masetero y pterigoideo medial

SUMMARY: Both the masseter and medial pterygoid muscles elevate the mandible, raising the lower jaw by acting simultaneously on the lateral and medial surfaces of the mandibular ramus. Nevertheless, electromyographic studies indicate that these muscles, as well as the superficial and deep heads of the masseter, act in a different way during mastication. We have analyzed by real time quantitative polymerase chain reaction (RT-qPCR) the expression of myosin heavy chain (MHC) isoforms in the masseter and medial pterygoid muscles in humans in order to identify possible differences in the expression patterns that may be related to functional differences identified with electromyography. Our findings indicate that the expression pattern of MHC isoforms in the two muscles is characteristic of fast and powerful phasic muscles. We have also observed a high percentage of expression of the MHC-IIx isoform and the expression of the MHC-M isoform at the mRNA level in both muscles, an isoform that does not translate into protein in the masticatory muscles of humans. The high percentage of expression of the MHC-IIx isoform in humans can be related to a high contractile speed of the masseter and medial pterygoid in humans. On the other hand, the low percentage of expression of the MHC-M isoform at the mRNA level in both muscles can be related to the complex evolutionary process that has reduced the size and force of the masticatory muscles in humans.

RESUMEN: Los músculos masetero y pterigoideo medial elevan la mandíbula actuando de forma simultánea sobre las caras lateral y medial de su rama. Sin embargo, los estudios electromiográficos indican que estos dos músculos actúan de forma diferente durante la masticación, de la misma forma que lo hacen las porciones superficial y profunda del músculo masetero. En el presente estudio hemos analizado mediante PCR en tiempo real la expresión de las isoformas de la cadena pesada de la miosina o myosin heavy chain (MHC) en los músculos masetero y pterigoideo medial en humanos, con la finalidad de identificar diferencias en los patrones de expresión que se puedan relacionar con las diferencias funcionales identificadas con la electromiografía. Nuestros resultados indican que el patrón de expresión de las isoformas de la MHC en los dos músculos es la característica de los músculos rápidos y potentes. También hemos observado un elevado porcentaje de expresión de la isoforma MHC-IIx y la expresión a nivel de ARNm de la isoforma MHC-M en los dos músculos, una isoforma que no se detecta a nivel de proteína en los músculos masticadores humanos. El elevado porcentaje de expresión de la isoforma MHC-IIx que hemos observado se puede relacionar con una elevada velocidad de contracción de los músculos masetero y pterigoideo medial en los humanos. Por otro lado, el bajo porcentaje de expresión de la isoforma MHC-M a nivel de ARNm en ambos músculos se puede relacionar con los procesos evolutivos complejos que han reducido el tamaño y la fuerza de los músculos masticadores en los humanos.

Differential expression of Dp71 and Dp40 isoforms in proliferating and differentiated neural stem cells: Identification of Dp40 splicing variants.

Dp71 and Dp40 are the main products of the DMD gene in the central nervous system, and they are developmentally regulated from the early stages of embryonic development to adulthood. To further study the roles of Dp71 and Dp40 during cell proliferation and neural differentiation, we analyzed Dp71/Dp40 isoform expression at the mRNA level by RT-PCR assays to identify alternative splicing (AS) in the isoforms expressed in rat neural stem/progenitor cells (NSPCs) and in differentiated cells (neurons and glia). We found that proliferating NSPCs expressed Dp71d, Dp71dΔ71, Dp71f, Dp71fΔ71, Dp71dΔ74 and Dp40, as well as two Dp40 isoforms: Dp40Δ63,64 and Dp40Δ64-67. In differentiated cells we also found the expression of Dp71d, Dp71dΔ71, Dp71f, Dp71fΔ71 and Dp40. However, the expression frequencies were different in both stages. In addition, in differentiated cells, we found Dp71fΔ71-74, and interestingly, we did not find the expression of Dp71dΔ74 or the newly identified Dp40 isoforms. In this work we show that NSPC differentiation is accompanied by changes in Dp71/Dp40 isoform expression, suggesting different roles for these isoforms in NSPCs proliferation and neuronal differentiation, and we describe, for the first time, alternative splicing of Dp40.

A comprehensive analysis of AHRR gene as a candidate for cleft lip with or without cleft palate.

Cleft lip and palate (CL/P) is among the most common congenital malformations and affects 1 in 700 newborns. CL/P is caused by genetic and environmental factors (maternal smoking, alcohol or drug use and others). Many genes and loci were associated with cleft lip/palate but the amount of heterogeneity justifies identifying new causal genes and variants. AHRR (Aryl-Hydrocarbon Receptor Repressor) gene has recently been related to CL/P however, few functional studies analyze the genotypephenotype interaction of AHRR with CL/P. Several studies associate the molecular pathway of AHRR to CL/P which indicates this gene as a functional candidate in CL/P etiology. METHODS: Systematic Literature Review was performed using PUBMED database with the keywords cleft lip, cleft palate, orofacial cleft, AHRR and synonyms. SLR resulted in 37 included articles. RESULTS: AHRR is a positional and functional candidate gene for CL/P. In silico analysis detected interactions with other genes previously associated to CL/P like ARNT and CYP1A1. AHRR protein regulates cellular toxicity through TCDD mediated AHR pathway. Exposure to TCDD in animal embryos is AHR mediated and lead to cleft palate due to palate fusion failure and post fusion rupture. AHRR regulates cellular growth and differentiation, fundamental to lip and palatogenesis. AHRR decreases carcinogenesis and recently a higher tumor risk has been described in CL/P patients and families. AHRR is also a smoking biomarker due to changed methylation sites found in smokers DNA although folate intake may partially revert these methylation alterations. This corroborates the role of maternal smoking and lack of folate supplementation as risk factors for CL/P. CONCLUSION: This research identified the importance of AHRR in dioxin response and demonstrated an example of genetic and environmental interaction, indispensable in the development of many complex diseases.

Expression analysis of ST3GAL4 transcripts in cervical cancer cells.

ST3GAL4 gene expression is altered in different cancer types, including cervical cancer. Several mRNA transcripts have been reported for this gene; however, their expression levels in cervical cancer have not been analyzed. ST3GAL4 encodes for ß­galactosidase α­2,3­sialyltransferase 4, involved in the biosynthesis of the tumour antigens sLe(x) and sulfo­sLe(x). The present study evaluated the presence of three mRNA variants (V1, V2 and V3) in cervical cancer cell lines, detecting the three variants. Additionally, the expression level of the V1 transcript of the ST3GAL4 gene was determined by reverse transcription­quantitative polymerase chain reaction in cervical cell lines and in normal, premalignant and cervical cancer tissue. The V1 transcript of the ST3GAL4 demonstrated significant decreased expression in premalignant and malignant cervical tissues. The results suggested that deregulation of this gene could occur prior to the presence of cancer and demonstrated the importance of evaluating the expression level of V1, and its association with disease progression.

Oestrogen receptor beta isoform expression in sporadic colorectal cancer, familial adenomatous polyposis and progressive stages of colorectal cancer.

BACKGROUND: Among the sex hormones, oestrogen may play a role in colorectal cancer, particularly in conjunction with oestrogen receptor-ß (ERß). The expression of ERß isoform variants and their correlations with familial adenomatous polyposis (FAP) syndrome and sporadic colorectal carcinomas are poorly described. METHODS: This study aimed to investigate the expression levels of the ERß1, ERß2, ERß4 and ERß5 isoform variants using quantitative RT-PCR (921 analyses) in FAP, normal mucosa, adenomatous polyps and sporadic colorectal carcinomas. RESULTS: Decreased expression of ERß isoforms was identified in sporadic polyps and in sporadic colorectal cancer as well as in polyps from FAP syndrome patients compared with normal tissues (p < 0.001). In FAP patients, ERß1 and ERß5 isoforms showed significant down-expression in polyps (p < 0.001) compared with matched normal tissues. However, no differences were observed when sporadic colorectal carcinomas were compared to normal mucosa tissues. These findings suggest an association of the ERß isoform variants in individuals affected by germline mutations of the APC gene. Progressively decreased expression of ERß was found in polyps at early stages of low-grade dysplasia, followed by T1-T2 and T3-T4 tumours (p < 0.05). In sporadic colorectal cancer, the loss of expression was an independent predictor of recurrence, and ERß1 and ERß5 expression levels were associated with better disease-free survival (p = 0.002). CONCLUSION: These findings may provide a better understanding of oestrogens and their potential preventive and therapeutic effects on sporadic colorectal cancer and cancers associated with FAP syndrome.

Chronic low-dose exposure to a mixture of environmental endocrine disruptors induces microRNAs/isomiRs deregulation in mouse concomitant with intratesticular estradiol reduction.

Humans are environmentally exposed not only to single endocrine-disrupting chemicals (EDCs) but to mixtures that affect their reproductive health. In reproductive tissues, microRNAs (miRNAs) are emerging as key targets of EDCs. Here, we analysed changes in the testis "miRNome" (and their biogenesis mechanism) in chronically exposed adult mice to a cocktail of five EDCs containing 0.3 mg/kg-body weight (BW)/day of each phthalate (DEHP, DBP, BBP) and 0.05 mg/kg-BW/day of each alkylphenol (NP, OP), from conception to adulthood. The testis "miRNome" was characterised using next-generation sequencing (NGS). Expression levels of genes involved in miRNA biogenesis were measured by RT-qPCR, as well as several physiological and cytological parameters. We found two up-regulated, and eight down-regulated miRNAs and thirty-six differentially expressed isomiRs along with an over-expression of Drosha, Adar and Zcchc11. A significant decrease of intratesticular estradiol but not testosterone was detected. Functional analysis showed altered spermatogenesis, germ cell apoptosis and negative correlation of miR-18a-5p with Nr1h2 involved in the deregulation of the steroidogenesis pathway. Here, we present the first association between miRNA/isomiRs deregulation, their mechanisms of biogenesis and histopathological and hormonal alterations in testes of adult mice exposed to a mixture of low-dose EDCs, which can play a role in male infertility.

Identification of spliced mRNA isoforms of retinoid X receptor (RXR) in the Oriental freshwater prawn Macrobrachium nipponense.

Retinoid X receptors (RXR) are members of the nuclear receptor family that are conserved from invertebrates to vertebrates, and they play an essential role in regulating reproductive maturation, molting, and embryo development. In this study, five RXR isoforms, named RXRL2 (L, long form), RXRL3, RXRS1 (S, short form), RXRS2, and RXRS3, containing six domains from A to F, were cloned from the prawn Macrobrachium nipponense using 5'- and 3'- rapid amplification of cDNA ends. Differences among their structures were observed not only in the D and E domains but also in the A/B domain, which were previously found in insects but not in crustaceans. This is the first report to show that differences occur in the A/B domain of RXR in crustaceans. RXR expressions were also examined in various tissues including the ovary, testis, muscle, hepatopancreas, heart, gill, stomach, intestine, and cuticle. Expression pattern investigations indicated that the five isoforms were differentially expressed. RXRS3 was only detected in the ovary, and the other RXRs were abundant in the ovary and testis. These data suggested that RXR mediates a series of processes related to reproduction.

Mycobacterium bovis infection in cattle induces differential expression of prolactin receptor isoforms in macrophages.

Prolactin receptor (PRLr) is a member of the cytokine receptor superfamily 1 showing tissue specific structural diversity. Expression of PRLr isoforms in lymphoid tissues has been associated with immunomodulatory function of prolactin. Bovine tuberculosis (bTB) is characterized by chronic inflammation caused by the persistent infection of lymphoid tissues with Mycobacterium bovis. To test the hypothesis of the influence of PRLr in the pathogenesis of bTB, the aim of this study was to identify PRLr isoforms expressed during bTB in different tissues and to analyze their association with the pathogenesis of bTB. We examined lymphoid and non-lymphoid tissues ex vivo from experimentally and naturally infected cattle, as well as from bTB-free cattle, by Western blot (WB) and immunohistochemistry (IH). In vitro, monocytes from exposed, infected, and healthy cattle were stimulated with M. bovis antigens and then analyzed by WB. To detect transcriptional levels of PRLr in macrophages (MØ) exposed to M. bovis, real time PCR was performed. WB revealed diversity of PRLr isoforms in tissues from infected cattle but not in tissues from bTB-free cattle. PRLr isoforms 100 kDa 75, 50 and 40 were found expressed in tissues of animals infected with M. bovis, while only the short isoform of 40 kDa correlated with the immunopathology and ability to infect MØ. We confirmed the synthesis of PRLr mRNA in MØ after M. bovis exposure and propose that molecular pathogen patterns of M. bovis might modulate inflammation during bTB through expression of the PRLr isoform in MØ.

The use of alternative polyadenylation sites renders integrin ß1 (Itgb1) mRNA isoforms with differential stability during mammary gland development.

Integrins are heterodimeric cell-surface adhesion receptors that play a critical role in tissue development. Characterization of the full-length mRNA encoding the ß1 subunit (Itgb1) revealed an alternative functional cleavage and polyadenylation site that yields a new Itgb1 mRNA isoform 578 bp shorter than that previously reported. Using a variety of experimental and bioinformatic approaches, we found that the two Itgb1 isoforms are expressed at different levels in a variety of mouse tissues, including the mammary gland, where they are differentially regulated at successive developmental stages. The longer mRNA species is prevelant during lactation, whereas the shorter is induced after weaning. In 3D cultures, where expression of integrin ß1 protein is required for normal formation of acini, experimental blockade of the longer isoform induced enhanced expression of the shorter species which allowed normal morphological mammary differentiation. The short isoform lacks AU-rich motifs and miRNA target sequences that are potentially implicated in the regulation of mRNA stability and translation efficiency. We further determined that the AU-binding protein HuR appears to selectively stabilize the longer isoform in the mammary gland. In summary, the results of the present study identify a new regulatory instance involved in the fine-tuning of Itgb1 expression during mammary gland development and function.

Characterization of P1 promoter activity of the beta-galactoside alpha2,6-sialyltransferase I gene (siat 1) in cervical and hepatic cancer cell lines.

The level of beta-galactoside alpha2,6-sialyltransferase I (ST6Gal I) mRNA, encoded by the gene siat1, is increased in malignant tissues. Expression is regulated by different promoters - P1, P2 and P3 - generating three mRNA isoforms H, X and YZ. In cervical cancer tissue the mRNA isoform H, which results from P1 promoter activity, is increased. To study the regulation of P1 promoter, different constructs from P1 promoter were evaluated by luciferase assays in cervical and hepatic cell lines. Deletion of a fragment of 1048 bp (-89 to +24 bp) increased 5- and 3-fold the promoter activity in C33A and HepG2 cell lines, respectively. The minimal region with promoter activity was a 37 bp fragment in C33A cells. The activity of this region does not require the presence of an initiator sequence. In HepG2 cells the minimal promoter activity was detected in the 66 bp fragment. Sp1 (-32) mutation increased the promoter activity only in HepG2 cells. HNF1 mutation decreased promoter activity in HepG2 cell line but not in C33A cells. We identified a large region that plays a negative regulation role. The regulation of promoter activity is cell type specific. Our study provides new insights into the complex transcriptional regulation of siat1 gene.

Identification and characterization of microRNAs in Phaseolus vulgaris by high-throughput sequencing.

BACKGROUND: MicroRNAs (miRNAs) are endogenously encoded small RNAs that post-transcriptionally regulate gene expression. MiRNAs play essential roles in almost all plant biological processes. Currently, few miRNAs have been identified in the model food legume Phaseolus vulgaris (common bean). Recent advances in next generation sequencing technologies have allowed the identification of conserved and novel miRNAs in many plant species. Here, we used Illumina's sequencing by synthesis (SBS) technology to identify and characterize the miRNA population of Phaseolus vulgaris. RESULTS: Small RNA libraries were generated from roots, flowers, leaves, and seedlings of P. vulgaris. Based on similarity to previously reported plant miRNAs,114 miRNAs belonging to 33 conserved miRNA families were identified. Stem-loop precursors and target gene sequences for several conserved common bean miRNAs were determined from publicly available databases. Less conserved miRNA families and species-specific common bean miRNA isoforms were also characterized. Moreover, novel miRNAs based on the small RNAs were found and their potential precursors were predicted. In addition, new target candidates for novel and conserved miRNAs were proposed. Finally, we studied organ-specific miRNA family expression levels through miRNA read frequencies. CONCLUSIONS: This work represents the first massive-scale RNA sequencing study performed in Phaseolus vulgaris to identify and characterize its miRNA population. It significantly increases the number of miRNAs, precursors, and targets identified in this agronomically important species. The miRNA expression analysis provides a foundation for understanding common bean miRNA organ-specific expression patterns. The present study offers an expanded picture of P. vulgaris miRNAs in relation to those of other legumes.