The Structure of the Gene for miR-223 in Humans, a MicroRNA Highly Expressed in Hematopoietic Tissues.

MicroRNAs (miRNA) are highly conserved noncoding regulatory RNA found abundantly in multicellular organisms. They derive from long primary transcripts (pri-miRNA) and act in a variety of regulatory pathways, including cell proliferation, apoptosis, fat metabolism and organogenesis. Over 200 human miRNA were identified by computational analysis and little is known about the gene hosts. Thus far, only 4 genes specific for microRNA have been characterized. Here we describe the structure of the gene for miR-223, a microRNA highly abundant in hematopoietic tissues. One cluster among 21,134 ESTs that we obtained from human bone marrow matched GenBank CD109334. Sequencing of cDNA from normal and leukemic cells did not correspond to known or potential protein structure, and revealed two alternative forms, with two or three “exons”. Phylogenetic analysis showed that our 234-nucleotide (nt) sequence matched DNA sequences of pri-miRNA of 6 non-human primates, with absolute identity in a 38-nt region that encompasses the 21-nt of the mature form. Secondary structure analysis of the transcribed RNA using M-Fold showed the stem-loop structure characteristic of a pri-miRNA. Northern blot with a probe designed to span the three exons of the genome sequence confirmed the expression of multiple bands in normal bone marrow, spleen, and acute and chronic leukemias, whereas other tissues were negative. Real time PCR with specific probes demonstrated that the expression of both alternate forms was restricted to the same cells. Northern blot revealed that mature miR-223 was observed only in hematopoietic cells. Thus, we describe for the first time the structure of a human non protein-coding gene specific for miRNA, spanning 4,450 nt of the genome in chromosome Xq12, with two transcript isoforms with 1,096 and 971 nt respectively, comprising three exons with 27, 125 and 944 nt, and show that the mature mRNA derives from the 3rd exon. Finally, there is an inverse relationship between the abundance of primary and mature species of miR-223 in normal and leukemic hematopoietic tissues, probably owing to a balance between transcription and processing of primary miRNA.