Conservation of miR-15a/16-1 and miR-15b/16-2 clusters

Abstract

MiR-15a/16-1 and miR-15b/16-2 clusters have been shown to play very important roles in regulating cell proliferation and apoptosis by targeting cell cycle proteins and the antiapoptotic Bcl-2 gene. However, the physiological implications of those two clusters are largely elusive. By aligning the primary miR-15a/16-1 sequence among 44 vertebrates, we found that there was a gap in the homologous region of the rat genome. To verify that there was a similar miR-15a/16-1 cluster in rats, we amplified this region from rat genomic DNA using PCR and found that a 697-bp sequence was missing in the current rat genome database, which covers the miR-15a/16-1 cluster. Subsequently, we also investigated the expression pattern of individual miRNAs spliced from miR-15a/16-1 and miR-15b/16-2 clusters, including miR-15a, miR-15a*, miR-15b, miR-15b*, miR-16-1/2, and miR-16-1/2* from various rat tissues, and found that all of those miRNAs were expressed in the investigated tissues. MiR-16 was most expressed in the heart, followed by the brain, lung, kidney, and small intestine, which indicates tissue specificity for individual miRNA expression from both clusters. Our results demonstrated that both miR-15a/16-1 and miR-15b/16-2 clusters are highly conserved among mammalian species. The investigation of the biological functions of those two clusters using transgenic or knockout/knockdown models will provide new clues to understanding their implications in human diseases and finding a new approach for miRNA-based therapy.

Fig. 1

Conservation of miR-15a/16-1 and miR-15b/16-2 clusters. a The miR-15a/16-1 cluster is highly conserved among mammalian species except that it is missing in the rat genome database. b The sequence of the miR-15b/16-2 cluster was aligned among 44 vertebrates and showed conservation

Fig. 2

Alignment of mature miRNAs spliced from miR-15a/16-1 and miR-16-2 clusters. Mature miRNA sequences from miR-15a, 15a*, 15b, 15b*, 16, and 16* were aligned among humans, mice, and rats. The different nucleotides are indicated in underlined italics

Fig. 3

miR-15a/16-1 and miR-15b/16-2 host genes. a The miR-15a/16-1 cluster is in the reverse strand and has the same transcriptional orientation as the host gene DLEU2. There are eight different transcripts of DLEU2, none of which encodes proteins. b The miR-15b/16-2 cluster is in the forward strand and has the same transcriptional orientation as the host gene SMC4. There are four different transcripts of SMC4, all of which encode protein

Fig. 4

Secondary structure of rat miR-15a/16-1. a The secondary structure of rat pre-miR-15a was predicted using the RNA MFOLD program. The mature miR-15a sequence is displayed in italics; the miR-15* sequence is underlined. b The secondary structure of rat pre-miR-16-1 was predicted using the RNA MFOLD program. MiR-16 is shown in italics; miR-16* is underlined

Fig. 5

The expression of individual miRNAs spliced from the miR-15a/16-1 and miR-15b/16-2 clusters was detected using polyA tailing RT-PCR from eight different tissues of 2-month-old Sprague-Dawley rats: 1, miR-15a; 2, miR-15a*; 3, miR-15b; 4, miR-15b*; 5, miR-16; 6, miR-16*; 7, RT negative control. U6 noncoding RNA was also amplified as internal controls from different tissues. For RT negative control, the reverse transcription was run simultaneously with other samples without the reverse transcriptase. PCR was performed by detecting only miR-16 from each tissue; no PCR product was visualized from agarose gel. The template for negative control of U6 was selected from heart