The purpose of this study was to investigate the alternative splicing in equine cordon-bleu WH2 repeat protein-like 1 (COBLL1) gene that was identified in horse muscle and blood leukocytes, and to predict functional consequences of alternative splicing by bioinformatics analysis. In a previous study...
The purpose of this study was to investigate the alternative splicing in equine cordon-bleu WH2 repeat protein-like 1 (COBLL1) gene that was identified in horse muscle and blood leukocytes, and to predict functional consequences of alternative splicing by bioinformatics analysis. In a previous study, RNA-seq analysis predicted the presence of alternative spliced isoforms of equine COBLL1, namely COBLL1a as a long form and COBLL1b as a short form. In this study, we validated two isoforms of COBLL1 transcripts in horse tissues by the real-time polymerase chain reaction, and cloned them for Sanger sequencing. The sequencing results showed that the alternative splicing occurs at exon 9. Prediction of protein structure of these isoforms revealed three putative phosphorylation sites at the amino acid sequences encoded in exon 9, which is deleted in COBLL1b. In expression analysis, it was found that COBLL1b was expressed ubiquitously and equivalently in all the analyzed tissues, whereas COBLL1a showed strong expression in kidney, spinal cord and lung, moderate expression in heart and skeletal muscle, and low expression in thyroid and colon. In muscle, both COBLL1a and COBLL1b expression decreased after exercise. It is assumed that the regulation of COBLL1 expression may be important for regulating glucose level or switching of energy source, possibly through an insulin signaling pathway, in muscle after exercise. Further study is warranted to reveal the functional importance of COBLL1 on athletic performance in race horses.
The purpose of this study was to investigate the alternative splicing in equine cordon-bleu WH2 repeat protein-like 1 (COBLL1) gene that was identified in horse muscle and blood leukocytes, and to predict functional consequences of alternative splicing by bioinformatics analysis. In a previous study, RNA-seq analysis predicted the presence of alternative spliced isoforms of equine COBLL1, namely COBLL1a as a long form and COBLL1b as a short form. In this study, we validated two isoforms of COBLL1 transcripts in horse tissues by the real-time polymerase chain reaction, and cloned them for Sanger sequencing. The sequencing results showed that the alternative splicing occurs at exon 9. Prediction of protein structure of these isoforms revealed three putative phosphorylation sites at the amino acid sequences encoded in exon 9, which is deleted in COBLL1b. In expression analysis, it was found that COBLL1b was expressed ubiquitously and equivalently in all the analyzed tissues, whereas COBLL1a showed strong expression in kidney, spinal cord and lung, moderate expression in heart and skeletal muscle, and low expression in thyroid and colon. In muscle, both COBLL1a and COBLL1b expression decreased after exercise. It is assumed that the regulation of COBLL1 expression may be important for regulating glucose level or switching of energy source, possibly through an insulin signaling pathway, in muscle after exercise. Further study is warranted to reveal the functional importance of COBLL1 on athletic performance in race horses.
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문제 정의
Despite various studies on COBLL1, the specific roles of both genes are still unclear, especially in horses. In this study, we focused on the analysis of the COBLL1 gene which was one of the alternatively spliced genes during exercise. We confirmed alternative splicing forms of COBLL1 in various horse tissues and examined the expression pattern of each alternative splicing form in response to exercise.
제안 방법
Given the importance of glucose metabolism in exercise, it is of interest that the regulation of COBLL1 expression maybe important for regulating glucose level or switching of energy source, possibly through an insulin signaling pathway, in muscle after exercise. Taken together, this information may be helpful to investigate the role of equine COBLL1 on the metabolic changes related to the athletic performance of racehorses and to develop COBLL1 as a useful biomarker for athletic performance.
To analyze the expression level of COBLL1alternative splicing isoforms in muscle before and after exercise, realtime quantitative polymerase chain reaction (RT-qPCR) was conducted by using the BioRad CFX-96 machine (BioRad, Hercules, CA, USA). Primer sequences were designed to detect each type of alternative splicing forms.
Two primer pairs, Cobll1a-S (5’-ATCAGTAGACCCGCACGGAA-3’) and Cobll1a-AS (5’- ACACTGACGGAGGAAACCTC-3’) for COBLL1a, and Cobll1b-S (5’-AGGGTCTGTCCAGCCCAGCT-3’) and Cobll1b-AS (5’- CCATAACGTGTGGTCCCTGTCC-3’) for COBLL1b, were used to specifically detect each isoform.
대상 데이터
Three male Thoroughbred horses aged 5, 9, and 10 years, which were maintained at Ham-an Racing Horse Resort and Training Center, were used to obtain the blood and skeletal muscle samples before and after exercise. Exercise was performed by trotting at the speed of 13 km/h for 30 min.
이론/모형
All samples were measured in triplicate to ensure reproducibility, and Ct value was used to calculate the fold change by using the 2–ΔΔCt method (Livak and Schmittgen, 2001).
2). The amino acid sequences were aligned and used for generating phylogenetic tree by using the MUSCLE and Neighbor-Joining methods, respectively, in MEGA6 program (Tamura et al., 2013). The web-based protein prediction program, PredictProtein (www.
(B) Phylogenetic tree of COBLL1. The phylogenetic tree was made with the full amino acid sequence of each species by Neighbor-Joining method after aligned by the MUSCLE method in MEGA6 program. Horse COBLL1 was similar to cow and dog while divergent from frog and chicken.
(A) Alignment of N-terminal amino acid sequences of COBLL1 from various species. The sequences were aligned by the MUSCLE method in MEGA6 program. The N-terminal sequences showed higher similarity among the species compared to C-terminal sequences.
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