The results also highlight the conserved role of miR-29c-3p across humans and mice to regulate RAG1 ( Figures 1B and S1G–S1I). The results suggest that although many miRNAs could be responsible for the regulation of RAG1 in various cell types and tissues, only miR-29c-3p expression inversely corelated to RAG1 at different stages of B cell development. Because miRNAs regulate gene expression by binding to the 3′ UTR of mRNAs, raw reads selected from RNA-seq datasets were mapped onto mm10 and hg19, mouse and human reference genomes, respectively, using Bowtie aligner, and mapped reads were analyzed ( Figure 1A). RAG expression was elevated in centrocytes and pre-GCB cells in human tonsillar tissue and low in plasma and naive B cell stages. Additional datasets of human (SRA: SRP002958), comprising centrocytes, pre-GCB (germinal center B) cells, plasma, and naive and memory B cells from NCBI-SRA, were evaluated ( Figures S1C–S1E). To do this, small RNA sequencing (RNA-seq) datasets of mouse (accession ID Sequence Read Archive : SRP002412), comprising pro-B, pre-B, immature, and mature B cells, were analyzed. Considering that expression of RAGs differs in a stage-dependent manner, profiling of differentially expressed miRNAs during B cell developmental stages was examined. miRNAs harboring seed sequence that can bind to the 3′ UTR of RAG1 were short listed from TargetScan and miRDB. Although the length of miRNA varied from 14 to 29 nt, the majority were 21–24 nt long ( Figures S1A and S1B). To investigate the role of miRNA in the regulation of RAGs, first we analyzed using miRBase the distribution and frequency of miRNAs with respect to their length. Thus, our results reveal the role of miRNA in the regulation of RAG1 and its relevance in cancer. A negative correlation of miR-29c and RAG1 levels is also observed in leukemia patients, suggesting the potential use of miR-29c as a biomarker and a therapeutic target. The miR-29c expression is inversely proportional to RAG1 in a B cell developmental stage-specific manner, and miR-29c null mice exhibit a reduction in mature B cells. Modulation of miR-29c levels leads to change in V(D)J recombination efficiency in pre-B cells. Various lines of experimentation, including CRISPR-Cas9 genome editing, demonstrate the target specificity and direct interaction of miR-29c to RAG1. ![]() We report the regulation of RAG1 through a microRNA (miRNA), miR-29c, in a B cell stage-specific manner in mice and humans. Recombination activating genes (RAGs), consisting of RAG1 and RAG2, are stringently regulated lymphoid-specific genes, which initiate V(D)J recombination in developing lymphocytes.
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