In silico prediction of short hairpin RNA and in vitro silencing of activin receptor type IIB in chicken embryo fibroblasts by RNA interference.

Gene silencing by RNA interference is extensively used reverse genetic approach to analyse the implications of any gene in mammalian systems. The silencing of the Activin type IIB receptor belonging to transforming growth factor beta superfamily has demonstrated increase in muscle growth in many species. We designed five short hairpin RNA constructs targeting coding region of chicken ACTRIIB. All the shRNAs were transfected into chicken embryo fibroblast cells and evaluated their silencing efficiency by real time PCR and western blotting. Initially the computational analysis of target region and shRNA constructs was undertaken to predict sequence based features (secondary structures, GC% and H-b index) and thermodynamic features (ΔGoverall, ΔGduplex, ΔGbreak-target, ΔGintra-oligomer, ΔGinter-oligomer and ΔΔGends). We determined that all these predicted features were associated with shRNA efficacy. The invitro analysis of shRNA constructs exhibited significant (P < 0.05) reduction in the levels of ACTRIIB at mRNA and protein level. The knock down efficiency of shRNAs varied significantly (P < 0.001) from 83% (shRNA 1) to 43% (shRNA 5). All the shRNAs up regulated the myogenic pathway associated genes (MyoD and MyoG) significantly (P < 0.05). There was significant (P < 0.05) up-regulation of IFNA, IFNB and MHCII transcripts. The ACTRIIB expression was inversely associated with the expression of myogenic pathway and immune response genes. The anti ACTRIIB shRNA construct 1 and 3 exhibited maximum knock down efficiency with minimal interferon response, and can be used for generating ACTRIIB knockdown chicken with higher muscle mass.

Molecular characterization and computational structure prediction of activin receptor type IIB in aseel and broiler chicken.

The present study was formulated to characterize and comprehend the molecular structural characteristics of ACTRIIB receptor in Aseel and control broiler (CB) populations. The full length coding sequence (1539 bp) of the receptor was amplified, cloned, sequenced and analyzed using bioinformatic tools. The physico chemical properties of protein and structural features like secondary structure, solvent accessibility and disorder regions were computed. The 3D structure was predicted by I-TASSER and evaluated by Ramachandran Plot and tools under Structural Analysis and Verification Server. The nucleotides differences between CB and Aseel were c. [156G > A; 210 T > C; 493C > T; c.520G > C; 665A > C; 686G > A; 937C > G; 1011A > C; 1130A > G; 1208 T > A; 1326 T > C; 1433 T > C]. The amino acid substitutions between CB and Aseel were p. [(Pro165Ser; Glu174Gln; Gln222Pro; Ser229Asn; His313Asp; Gln377Arg; Val403Asp; and Ile478Thr)]. While, the silent changes includes p. [(Lys53=; Glu71=; Leu337=; Asp442=)]. The molecular weight of mature protein was predicted to be 55.51 kDa and 57.80 kDa in Aseel and CB, respectively. The higher rank 3D model had a C-score of -1.60 in Aseel and - 1.41 in CB, while the estimated TM-score (0.54 ±â€¯0.14) and RMSD (5.8 ±â€¯1.2 Å) were found to be similar in Aseel and CB. Among the 512 residues, >90% were in favored region, 4.7% in allowed region and <1.5% in disallowed region in both Aseel and CB. The pattern of contact map was comparable in Aseel and CB. The Hydrogen bond plots of the Aseel and CB shared similar secondary structure pattern. The ACTRIIB protein was predicted to interact with ACVR1B, ACVR1C, INHBA, SMAD 1,2,5,7 & 9 and BMPR1A&B. Clustal and phylogenetic analysis implied that both the lines were closely related and formed a sub cluster with in avian cluster. The current research provides insights about structural and functional aspects of the receptor and also aids in understanding the evolutionary history of ACTRIIB.