Novel lncRNA lncFAM200B: Molecular Characteristics and Effects of Genetic Variants on Promoter Activity and Cattle Body Measurement Traits

Abstract

Skeletal muscle is one of the three major muscle types in an organism and has key roles in the motor system, metabolism, and homeostasis. RNA-Seq analysis showed that novel lncRNA, lncFAM200B, was differentially expressed in embryonic, neonatal, and adult cattle skeletal muscles. The main aim of this study was to investigate the molecular and expression characteristics of lncFAM200B along with its crucial genetic variations. Our results showed that bovine lncFAM200B was a 472 nucleotide (nt) non-coding RNA containing two exons. The transcription factor binding site prediction analysis found that lncFAM200B promoter region was enriched with SP1 transcription factor, which promotes the binding of myogenic regulatory factor MyoD and DNA sequence. The mRNA expression analysis showed that lncFAM200B was differentially expressed in embryonic, neonatal, adult bovine muscle tissues, and the lncFAM200B expression trend positively correlated with that of MyoG and Myf5 in myoblast proliferation and differential stages. To identify the promoter active region of lncFAM200B, we constructed promoter luciferase reporter gene vector pGL3-Basic plasmids containing lncFAM200B promoter sequences and transfected them into 293T, C2C12, and 3T3-L1 cells. Our results suggested that lncFAM200B promoter active region was from −403 to −139 (264 nt) of its transcription start site, covering 6 SP1 potential binding sites. Furthermore, we found a novel C-T variation, named as SNP2 (ERZ990081 in European Variation Archive) in the promoter active region, which was linked to the nearby SNP1 (rs456951291 in Ensembl database). The genotypes of SNP1 and combined genotypes of SNP1 and SNP2 were significantly associated with Jinnan cattle hip height. The luciferase activity analysis found that the SNP1-SNP2 haplotype CC had the highest luciferase activity, which was consistent with the association analysis result that the combined genotype CC-CC carriers had the highest hip height in Jinnan cattle. In conclusion, our data showed that lncFAM200B is a positive regulator of muscle development and that SNP1 and SNP2 could be used as genetic markers for marker-assisted selection (MAS) breeding of beef cattle.

Identification of novel alternative splicing of bovine lncRNA lncFAM200B and its effects on preadipocyte proliferation

“…The 3′ and 5′ rapid amplification of complementary DNA (cDNA) ends (RACE) experiments were performed to identify the transcript variants of the cattle lncFAM200B gene according to a previous study (Zhang et al, 2019). The Coding Potential Calculator (CPC) website and the vitro prokaryotic expression system pET‐28a vector were used to predict and identify the coding potential of the transcript variants (Zhang et al, 2019). The full‐length lncFAM200B transcript variants were amplified and cloned into the pcDNA3.1 (+) vector using Xho I and Hin dIII restriction enzymes and the In‐Fusion HD Cloning Kit (TaKaRa; Zhang et al, 2019).…”

“…The Coding Potential Calculator (CPC) website and the vitro prokaryotic expression system pET‐28a vector were used to predict and identify the coding potential of the transcript variants (Zhang et al, 2019). The full‐length lncFAM200B transcript variants were amplified and cloned into the pcDNA3.1 (+) vector using Xho I and Hin dIII restriction enzymes and the In‐Fusion HD Cloning Kit (TaKaRa; Zhang et al, 2019).…”

“…The genotypic and allelic frequencies, Hardy–Weinberg equilibrium, heterozygosity, effective population size, and polymorphism information content (PIC) were calculated using the MSR website (http://www.msrcall.com/). The association analysis between the body measurement traits and the genotypes was performed using the reduced linear model: Y i = u + G i + e, according to a previous study (Zhang et al, 2019). The different breeds and different aged cattle were analyzed separately.…”

“…According to the sequencing results of the new bands, we designed primers to clone the full-length lncFAM200B. As a result, we obtained four kinds of transcript variants of bovine lncFAM200B (Figure 5), and the transcript variant 3 (lncFAM200B-V3) was the lncFAM200B identified in the previous study (Zhang et al, 2019). The full-length lncFAM200B-V1, lncFAM200B-V2, and lncFAM200B-V4 were 741, 572, and 780 bp, respectively ( Figure 5).…”

“…The primers used to detect the expression level of cattle lncFAM200B and the analysis method of the relative expression levels were the same as those in a published paper (Zhang et al, 2019). All samples were repeated three times.…”