Study Of The Affinity Of MiRNAs From The DLK1-GTL2 Imprinting Domain For The DLK1 Gene

DLK1-GTL2 imprinting domain plays important roles in embryonic development of multiple species, like human, mouse. An A to G mutation identified in this locus was thought to be the causative mutation of hindquarters muscular hypertrophy of Callipyge sheep. The heterozygous individuals only inheriting the CLPG mutation from their father express the hypertrophy phenotype, homozygous individuals inheriting the CLPG mutation from their father and mother is phenotypically "wildtype", this non-Mendelian mode of inheritance with parent-of-origin effect is called to be polar overdominance. It was thought Callipyge mutation developmentally regulates expression of genes at this locus in cis and in trans. DLK1 and PEG11 was regarded to be functional genes to result in muscular hypertrophy of Callipyge individuals. It has been proved that PEG11 was trans-inhibited by miRNAs processed from antiPEG11, the mechanism of trans-inhibition for DLK1 remains unclear. When compared to +mat/CLPGpat animals, DLK1 levels in skeletal muscle of CLPG/CLPG animals are downregulated at the protein (～10-fold) and at the mRNA (～3-fold) level. Considering that miRNAs generally modulate target gene expression at posttranscriptional level, miRNAs from DLK1-GTL2 imprinting domain become the first candidate to be invovled in trans-inhibition for DLKl. In this study, the affinity of miRNA from DLK1-GTL2 domain on DLK1 was evaluated on the basis of DLK1 protein quantifying, the main results are as follows:1. Empolying RACE technique, we identified the transcription start site (mapped to 174 bp upstream of the ATG start codon) and the predominant polyadenylation sites (236 bp downstream of stop codon TGA) of DLK1 C2 isoform which is most abundantly expressed in skeletal muscle.2. We generated the 1405 bp full-length "DLK1", introduce it to a slightly modified pcDNA3.1(-) vector, the resulting construct (p3.1M-DLK1-HA) was utilized to study of affinity of miRNA on DLK1.3. On the basis of small RNA sequencing data from longissimus dorsi of Callipyge animals, we designed 121 mimic miRNAs used for this study.4. We cotransfected COS1 cells with a mixture of DLK1-expressing p3.1M-DLK1-HA vector, GFP-expressing pcDNA3-GFP vector (for transfection efficiency correction), and individual mimic miRNA. Of the 121 miRNA mimics used for the evaluation of miRNAaffinity on DLK1, miR-329a-3p caused a 1.25-fold reduction (p= 3.8×10-4) in DLK1 levels when compared to the average of the negative controls. MiR-329b-3p, which differs from miR-329a-3p at only two residues near the 3’end, reduced DLK1 amounts 1.1-fold when compared to the overall average (p= 0.047). Seed matches of miR-329a-3p and miR-329b-3p in the full-length of DLK1. In addition, miR-329a-3p and miR-329b-3p accounted respectively for 0.0014% and 0.19% of miRNA molecules derived from the DLK1-GTL2 domain.5. Based on the study of the effects of the 121 tested miRNAs on DLK1, although the effect of miR-329a-3p is significant, we consider the evidence insufficient to claim that it is biologically relevant. Indeed, (ⅰ) the effects of the 121 tested miRNAs are approximately normally distributed, encompass the effects of the two negative controls, and are not correlated with predicted affinity for DLK1, (ⅱ) there are more miRNAs that have a significant positive effect than miRNAs that have a negative effect on DLK1 expression (which is unlikely to reflect a genuine biological activity given the known modus operandi of miRNAs), (ⅲ) the effect of miR-329a-3p becomes insignificant when considered jointly with the closely related miR-329b-3p, (ⅳ) the expression level of miR-329a-3p in ovine skeletal muscle is extremely low. What is certain is that none of the tested miRNAs is capable-under the utilized conditions- to single-handedly downregulate DLK1 to the extent that is observed in vivo in skeletal muscle of CLPG/CLPG sheep.Although a negative result was shown in this study, it provides reference data for the trans regulatory molecular mechanism of DLK1 and support for the study of molecular mechanism of Callipyge phenotype. For the first time, high-throughput protein quantifying assay was utilized for the study of interaction of multiple miRNAs and a particular target gene. It turned out none of these miRNAs was biologically related to predicted affinity to DLK1. This implies more complicated molecular mechanism is involved in Callipyge phenotype.