Cloning and expression profiling of testis-expressed microRNAs

Abstract

Using a new small RNA cloning method, we identified 141 miRNAs from the mouse testis, of which 29 were novel. The 141 miRNAs were mapped onto all chromosomes except the Y chromosome and 2/3 of these miRNA genes exist as clusters. approximately 70% of these miRNA genes were located in intronic or intergenic regions, whereas the remaining miRNAs were derived from exonic sequences. We further validated these cloned miRNAs by examining their expression in multiple mouse organs including developing testes and also in purified spermatogenic cells using semi-quantitative PCR analyses. Our expression profiling assays revealed that 60% of the testis-expressed miRNAs were ubiquitously expressed and the remaining are either preferentially (35%) or exclusively (5%) expressed in the testis. We also observed a lack of strand selection during testicular miRNA biogenesis, characterized by paired expression of both the 5' strands and 3' strands derived from the same precursor miRNAs. The present work identified numerous miRNAs preferentially or exclusively expressed in the testis, which would be interesting targets for further functional studies.

Fig. 1

Cloning of miRNAs from the mouse testis. (A) PCR products amplified using small RNA cDNAs of brain, the developing [postnatal day 7 (P7), P14, and P21] and adult testes. The lower bands with the expected size (∼120 nt) of miRNAs (arrow) was gel-extracted and subcloned for sequencing. A DNA ladder on each side indicates the size of the fragments. NTC stands for non-template control. (B) Size distribution of 141 miRNAs cloned from mouse testes aged P21 and adult. The miRNAs range from 20 to 24 nt in length, with 22 nt being the predominant size.

Fig. 2

Chromosomal distribution of miRNA genes and miRNA gene clusters identified in this study. (A) Chromosomal distribution of the 141 miRNA genes in the mouse genome. (B) Chromosomal distribution of 19 miRNA gene clusters and 28 miRNA genes exclusively or preferentially expressed in the testis. The mouse chromosomes were drawn to scale and aligned by their centromere position. Nineteen miRNA clusters (miRcs1−19) are indicated on the left and the 28 testis-specific or testis-preferential miRNAs are marked on the right. The miRc-19 genomic fragment containing the 11 X-linked testis-specific or testis-preferential miRNAs is enlarged and shown to the right of the X chromosome. The solid bars represent repetitive sequences and the open bars stand for the unique miRNA-coding sequences.

Fig. 3

Expression profiling for 122 miRNAs cloned from the mouse testis. Levels of miRNAs in 15 mouse tissues and two purified spermatogenic cells [pachytene (P.) spermatocytes and round (R.) spermatids] were determined using a semi-quantitative PCR analysis. (A) Expression profiles of mir-16, mir-t6 and mir-t25, representing the ubiquitous, preferential, and testis-specific expression patterns, respectively. A DNA ladder on each side indicates the size of the fragments. NTC stands for non-template control. (B) The heat map representing expression levels of the 122 miRNA in 15 mouse tissues and two purified spermatogenic cells [pachytene (P.) spermatocytes and round (R.) spermatids].

Fig. 4

Expression levels of 28 testis-specific or testis-preferential miRNAs in purified Sertoli cells and 5 spermatogenic cell populations including spermatogonia (Sg), pachytene spermatocytes (P), round spermatids (rS), elongated spermatids (eS) and vas spermatozoa (vS). (A) Three representative gel pictures showing expression levels of mir-t28, mir-t13 and mir-t3. A DNA ladder on each side indicates the size of the fragments. NTC stands for non-template control. (B) The heat map representing expression levels of the 28 testis-specific or testis-preferential miRNAs in purified Sertoli cells and 5 spermatogenic cell populations.

Fig. 5

Paired expression of miRNAs in the testis. The stem-loop structure of pre-miRNAs for three pairs of miRNAs [mir-t27/mir-741 (A), mir-742/mir-t23 (B), and mir-t25/mir-t24 (C)] are shown on the left and PCR amplicons of the 6 miRNAs (3 pairs) amplified from 15 mouse tissues and two purified spermatogenic cell populations (pachytene spermatocytes and round spermatids) are on the right. All of the 6 miRNAs were cloned from the testis and the four novel miRNAs are highlighted in gray in the stem-loop structures. The core RNA sequences used for designing PCR primers are indicated by arrows. The house-keeping miRNA mir-16 was used as a loading control. P7, P14 and P21: postnatal days 7, 14, and 21; P. Spermatocyte: pachytene spermatocyte; R. spermatid: round spermatid; A DNA ladder on each side indicates the size of the fragments. NTC stands for non-template control.