Figure 1.
Clone and sequence analysis of HongrES2 cDNA.
(A) Schematic representation of the 1.3-kb EST screened from a rat epididymal cDNA library, and the new 328 bp sequence obtained from two round 5′ RACE by a BD and Ambion kit, respectively. (B) Left panel: Northern blot analysis for HongrES2 with 32P-labeled 504-bp HongrES2 EST probe1 (seqNo861-1364) . E: epididymis; T: testis; 20 µg total RNA per lane; middle panel: Northern blot analysis for HongrES2 with 32P-labeled 1978 bp CES7 EST probe2 (seqNo116-2094); right panel: Northern blot analysis for HongrES2 with 32P-labeled 1191 bp CES7 EST probe3 (seqNo498-1689). (C) Upper panel: Chromosomal localization of the HongrES2 gene. The dark gray box is exon 1 from rat chromosome 5, and the light gray box is exon 2 from chromosome 19. The small white box represents the 7-nucleotide overlap from these two exons. e1, exon1; e2, exon2. Lower panel: A sequence alignment of CES7 and HongrES2. Twelve exons of the CES7 cDNA are depicted in the red rectangle. The same 216- bp fragment of the 3′ end of CES7 exon 12 and HongrES2 exon 2 is depicted by a purple shadow. The location of probe1, probe2 and probe3 for Northern blot analysis in figure1B is also marked out. (D) Left panel: RT-PCR validation using primers spanning the chimeric junction portion of HongrES2 cDNA. The product in lane1was got by the primer pairs of upper primer lane1 and common lower primer showed on the right panel. The product in lane2 was got by the primer pairs of upper primer lane2 and common lower primer; right panel: the schematic representation of the location of the two primer pairs used in the RT-PCR. Lane3: negative control; lane4: positive control. (E) Upper panel: ORF analysis of HongrES2 cDNA on line. Three short frames were displayed by rectangles and their sequential number and length were listed. Low panel: The full-length gene sequence of the HongrES2 cDNA. The gray box represents the poly(A) addition signal. The line labels the probe1 sequence used for the northern blot and in situ hybridization in Figure 3A.
Figure 2.
Regional and temporal expression of HongrES2 RNA.
(A) The tissue distribution of HongrES2 RNA. (B, C) The region-specific expression pattern, as shown by northern blot analysis, QRT-PCR ,and in situ hybridization. CP:caput; CO:corpus; CD: cauda; an 18S probe was used on the stripped membrane as an internal loading control. Bar = 2.0 mm. The primers used for RT-PCR ,Real-time PCR was listed in Table1. (D) The upper panel shows the northern blot analysis of HongrES2 RNA and 18S rRNA during development. The lower panel shows the relative amounts of HongrES2 RNA in the rat epididymis at different developmental stages. (E) The upper panel shows Northern blot analysis of adult rat epididymal HongrES2 RNA. level from pre-castration (d0) and castration for 1, 3, 5, and 7 days (d1, d3, d5, and d7) as well as for 1, 3, 5, and 7 days after the initial injection of testosterone propionate applied to the 7d-castrated rats (d7+1, d7+3, d7 +5, and d7 +7). Injections were continued every 2 days. The lower panel showed the relative expression levels of HongrES2 RNA (hybridization density of HongrES2 RNA/18S ribosomal RNA) in the rat.
Figure 3.
HongrES2 RNA is the precursor of a new miRNA-like small RNA.
(A) Panel 1: the in situ hybridization shows the subcellular localization of the 1.6-kb HongrES2 transcript in the cauda region. Panel 2: the in situ hybridization shows the cytoplasmic localization of the Bin1b transcript in the caput region as a control (100×); bar = 125 µm. Panel 3: the in situ hybridization shows the nuclear location of U6 RNA in the corpus region as a positive control (40×); bar = 200 µm. (B) The secondary structure prediction of the HongrES2 RNA by mfold. The predicted stem loop structure and mature sequence are labeled with purple and yellow, respectively. (C) A schematic representation of the different cDNA fragments described in (B). (D) Northern blot analysis showing three forms of the HongrES2 RNA in the rat epididymis. CD: cauda; CO: corpus; CP: caput; marker: Ambion small RNA marker (10 nt–100 nt). The marker lane was exposed for a shorter time on the same membrane. The blastn search result of the mil-HongrES2 sequence against the miRBase database. (E) Upper panel: depiction of how the mil-HongrES2 was bailed from the small RNA library by selective primers (GSP&3′adaptor primer). Low pannel: the mil-HongrES2 sequences identified from the small RNA library and the miRNA:miRNA* like duplex sequence. Real-time PCR showed the camparative quantitation of the mil-HongrES2 molecules in the whole small RNA library. (F) HongrES2 is processed into mil-HongrES2 in PC1 cells. Lane1: RNA from PC1 cells transfected with the HongrES2 expression vector. Lane2: The 25 nt RNA oligo nucleotide used as a positive control and the size marker for the processed product. The strong band below pre form of mil-HongrES2 pointed out by arrow was analysed through poly(A) tailing PCR using the gene specific forward primer. (G) Northern blotting to detect mil-HongrES2 after anti-FLAG-Ago2 immunoprecipitation. The right panel shows PC1 cells that were transfected with HongrES2 expression plasmids alone (mock) or in combination with plasmids that express FLAG-tagged Argonaute2 (Flag-Ago2). The left panel shows a control experiment using a probe of a known mir-29a to demonstrate that the IP worked. (H) The left panel shows that the Dicer protein level was reduced by two different siRNAs. The right panel shows that mil-HongrES2 expression was inhibited in DCR knockdown cells. Mir-29a expression was also detected as a control to show that the whole RNAi experiment was effective. Nosi: control siRNA. siDCR1/2: two different sequences of the siRNA-targeting mouse DCR gene. (I) Upper panel :sequence alignment of the 100-bp conserved sequence between different species of rat, mouse, and human around the mil-HongrES2 encoding region. Red rectangles labeled out the mil-HongrES2 sequence. Lower panel: northern blot analysis of mouse mil-HongrES2 expression, using the rat LNA probe of mil-HongrES2 (24 nt). Hybridization was carried out at 42°C.
Figure 4.
Over-expression of mil-HongrES2 caused by inflammation of the rat epididymitis.
(A) The upper panel showed Northern blot analysis of the RNA expression of HongrES2.18S rRNA was used as the loading control. The lower panel was the Real-time PCR analysis of Ncp2 besides HongrES2. (B,C) Northern blot analysis to detect the mil-HongrES2 expression. U6 probe was used on the stripped membrane as an internal loading control.
Figure 5.
CES7 was down-regulated by HongrES2 via mil-HongrES2.
(A) Western blot indicating the amount of CES7 protein after co-transfection into PC1 cell with the CES7 expression vector and different constructs. Mock: pcmv-tag4 plasmid without insertions. H2: Pcmv-tag4a-H2. H2T: Pcmv-tag4a-H2T. (B) Western blot indicating the amount of CES7 protein after co-transfection into PC1 cell with the CES7 expression vector and dsRNA regulators. Nosi1 and Nosi2: negative control of unrelated dsRNAs. (C) Depiction of the wide-type and the target-site mutant 3′UTR of CES7 cDNA inserted into the PRL-TK luciferase reporter vector. (D) Dual luciferase assay activity. H2: pcmv-tag4a-H2; H2-2: plenty-H2-2; H2T: pcmv-tag4a-H2T; A+1: the imperfect duplex of mil-HongrES2 mimics; D+1: the perfect duplex of mil-HongrES2 mimics; the data are expressed as the mean ± SEM (N>3). **P<0.01, ***P<0.001. All of the plasmids and mimics are shown in Table 2. (E) Expression of CES7 mRNA and HongrES2 RNA during the lifespan of the rat epididymis.
Figure 6.
Over-expression of mil-HongrES2 by injecting agomir into the epididymis to reduce the cauda sperm capacitation.
(A) Mil-HongrES2 over-expression by mil-HongrES2 mimics agomir down-regulation of CES7 protein expression. The two upper panels show the northern blot analysis of mil-HongrES2 over-expression. C: control group. H2: over expression group. U6 was used as the internal loading control. The number shows two or three different individuals. The two lower panels show the corresponding CES7 protein level in vivo, and α-tubulin was used as the loading control. The experiments were carried out independently and the results of 3 replicates are shown and indicated by the replicate number (1st, 2nd or 3rd). (B) Quantification analysis of mil-HongrES2 over-expression and the down-regulation of CES7 protein from (A) (n = 7 ). The data are expressed as the mean ± SEM. **P<0.001. (C) The change in protein tyrosine phosphorylation in mil-HongrES2 over-expressing sperm after incubation in the sperm culture medium. Total protein from the spermatozoa was collected for western blot analysis after 2 h, 3 h and 4 h of incubation; α-tubulin was used as the loading control.