Fig 1: Identification of piRNAs in human primary CD4 T-lymphocytes. (A) Deep sequencing shows the distribution of small RNAs in the activated CD4 T—lymphocytes. The arrows indicate piRNA peaks. The length cutoff is 18–40 bp. (B) Sequences of the abundant piRNAs. Selected piRNAs were annotated in GenBank. (C) Sequence alignment analysis of specific snoRNA-derived piRNAs. Conserved boxes are highlighted. (D) Schematic demonstration that the piRNA (piR30840) is derived from snoRNA. Cleavage sites are indicated by arrows. (E) One highly abundant piRNA (piR30840) was analyzed by northern blotting using a piR30840 antisense probe. (F) RNA pull-down assay using a piRNA, a scrambled sequence or a miRNA. The differential bands (indicated by arrows) were excised for mass spectrometry analysis. The locations of piRNA-associated Piwil4 and Ago4 are indicated by arrows. (G) Co-immunoprecipitation of Piwil4 with piRNA. The Piwil4-expressing plasmid was transfected into the PBMCs. HA-tagged Piwil4 was immunoprecipitated, and the associated piRNA was analyzed by qRT-PCR, with miR-181a as a control. The data represents three independent experiments. (H) Detection of 2′-O-methylation at the 3′-ends of piRNA by RTL-P. RT-PCR products were generated by using an unanchored (MeUA-RT) (1) or anchored (MeA-RT) RT primer (2) in the RT reaction at different concentrations of dNTPs. The data showed the average value of three independent experiments (right panel). (I) Expression of piR30840 in human CD4 T-lymphocytes, CD8 T-lymphocytes, B cells or monocytes. (J) Deep-sequencing reads of piR30840 compared to various miRNAs in the CD4 T lymphocytes.
Fig 2: TRAMP complex and exosome are involved in the decay of IL-4 pre-mRNA in the nucleus. (A) s30840 was co-transfected with siRNAs specific for trf4, air or mtr4 into CD4 T lymphocytes, respectively. The IL-4 expression was analyzed by western blotting. The data represents three independent experiments. (B) The interaction between Piwil4 and Mtr4 in 293T cells was detected by co-immunoprecipitation. Actin was used as loading control. (C) Piwil4 interacting with Prp4 was detected by co-immunoprecipitation in 293T cells. (D) s30840 was co-transfected with siRNA specific for rrp6, rrp44 and rrp42 into primary CD4 T-lymphocytes. The expression of IL-4 was detected by western blotting. The data represents three independent trials.
Fig 3: piRNA-guided Piwil4 and Ago4 participate in IL-4 pre-mRNA decay. (A) Schematic showing the primers used in detecting il-4 pre-mRNA. The primers f2/r14 and f21r21 were used to detect IL-4 pre-mRNA. (B and C) The effect of piR30840 on the IL-4 pre-mRNA. Primers were specific to the intron and exon of IL-4 (Figure 4A). The IL-4 pre-mRNA was examined in the purified CD4 T lymphocytes from PBMCs (B). The construct pIL4 was co-transfected with s30840 or control into the 293T cells. IL4 pre-mRNA was detected via qRT-PCR (C) analysis. Statistical significance between two samples was determined by using the student's t-test. P < 0.05. The data represents three independent experiments. (D) The interaction between Piwil4 and Ago4 in 293T cells was detected by co-immunoprecipitation. Actin was used as loading input control. (E) The s30840 was co-transfected with piwil4- or ago4-specific siRNAs into CD4 T lymphocytes, respectively. IL-4 expression was detected by western blotting. The data represents three independent trials. (F) The Piwil4-expressing construct was co-transfected with the pIL4 or pIL4m construct into the 293T cells. The Piwil4-associated IL-4 pre-mRNA was detected by RIP-qPCR. Statistical significance between two samples was determined by using the student’ t-test. P < 0.01. The data represents three independent experiments. (G) The Piwil4-expressing construct was co-transfected into 293T cells with the pIL-4 construct, with or without s30840. Piwil4-associated IL-4 pre-mRNA was detected by RIP-qPCR. P < 0.01. The data represents three independent trials. (H) Schematic graph showing the tethering assay system. pIn2s-5BoxB contains five 19 nt BoxB hairpins in the intron of IL-4, which can potently interact with various λN-PIWI or λN-AGO fusion proteins. (I) The construct containing In2s-5BoxB was co-transfected with the construct piwil4/ λN-piwil4 or ago4/λN-ago4. The effect on IL-4 expression was analyzed with western blotting.
Fig 4: Validation of selected upregulated (A) and downregulated (B) piRNAs in PIWIL4-targeting iSAE cells following RSV infection.Radar plots depicting the expression profiles of twenty selected piRNAs in PIWIL4-targeting (Targ) versus non-targeting (NTarg) iSAE cells either infected with RSV for 15 hours (red shaded area) or uninfected (control) (blue shaded area). piRNA expression levels were measured by RT-qPCR and calculated as fold changes relative to NTarg control cells using the 2^−ΔΔCT method. Data are normalized to U6 small nuclear RNA and presented as mean ± SEM (n=2, from two independent experiments run in triplicate).
Fig 5: Detection and differential expression of piRNAs in PIWIL4-silenced iSAE cells following RSV infection.iSAE cells were transfected with either non-targeting or PIWIL4-targeting siRNA and total RNA was extracted from uninfected cells (Ctrl) or infected with RSV for 15 hours. Samples from one experiment done in triplicate were pooled and the number of differentially expressed genes (DEGs) were identified by piRNA array analysis. (A) Venn diagrams illustrating the distribution of piRNAs detected among the four experimental groups: non-targeting control (NTarg Ctrl), non-targeting RSV-infected (NTarg RSV), PIWIL4 targeting control (Targ Ctrl), and PIWIL4 targeting RSV-infected (Targ RSV) groups. Numbers within each section indicate piRNAs detected exclusively in one condition or shared between conditions. For NTarg cells, 975 piRNAs were detected only in RSV-infected samples, 247 only in controls, and 9,948 for both conditions. For Targ cells, 650 piRNAs were unique to RSV-infected samples, 566 unique to controls, and 9,908 were detected in both. (B) Bar graphs display the numbers of differentially upregulated (open bars) or downregulated (black bars) expressed piRNAs comparing PIWIL4 targeting versus non-targeting conditions within either the control or RSV-infected condition. Differential expressions were determined using a fold-change cutoff of FC ≥ 1.5.
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