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A SnoRNA-derived piRNA interacts with human interleukin-4 pre-mRNA and induces its decay in nuclear exosomes

PIWI interacting RNAs (piRNAs) are highly expressed in germline cells and are involved in maintaining genome integrity by silencing transposons. These are also involved in DNA/histone methylation and gene expression regulation in somatic cells of invertebrates. The functions of piRNAs in somatic cells of vertebrates, however, remain elusive. We found that snoRNA-derived and C (C′)/D′ (D)-box conserved piRNAs are abundant in human CD4 primary T-lymphocytes. piRNA (piR30840) significantly downregulated interleukin-4 (IL-4) via sequence complementarity binding to pre-mRNA intron, which subsequently inhibited the development of Th2 T-lymphocytes. Piwil4 and Ago4 are associated with this piRNA, and this complex further interacts with Trf4-Air2-Mtr4 Polyadenylation (TRAMP) complex, which leads to the decay of targeted pre-mRNA through nuclear exosomes. Taken together, we demonstrate a novel piRNA mechanism in regulating gene expression in highly differentiated somatic cells and a possible novel target for allergy therapeutics.

of the samples with corresponding rank order was shown in the parenthesis. b The expression values of PIWIL4 in all available samples were retrieved from NCBI GEO Profiles database (www.ncbi.nlm.nih.gov/geoprofiles). The corresponding IDs were shown in the table, and the number of related samples was shown in the parenthesis. The citation(s) and title of each GEO Profiles ID were shown in the supplemental excel file.
C. Table 3. The ID numbers in NCBI GEO Profiles database of PIWIL4 in mouse CD4+ T-cells.  (2) 76618870(1), 95420122(1) 80-89 (1) 95420122(1) 90-100 (2) 75565738 (2) a The values shown in the column of Rank represent the percentile rank within the sample labeled on the x-axis in Figure 2. The number of the samples with corresponding rank order was shown in the parenthesis. b The expression values of PIWIL4 in all available samples were retrieved from NCBI GEO Profiles database (www.ncbi.nlm.nih.gov/geoprofiles). The corresponding IDs were shown in the table, and the number of related samples was shown in the parenthesis. The citation(s) and title of each GEO Profiles ID were shown in the supplemental excel file.

Supplementary Figure 2. Gene expression profiles of PIWIL4 in mouse/human CD4+ T-cells
The relative expression levels of all genes within a mRNA chip sample were rank ordered, and then split into 10 groups (1-9: lowest; 90-100: highest) shown on the x-axis. The percentile rank gives an indication of the relative expression level of PIWIL4 among all genes. The values shown on the y-axis represent the percentages of the mRNA genechip sample numbers with various ranked PIWIL4 expression within the total mRNA genechip sample numbers of mouse (n= 431) or human (n= 139) CD4+ T-cells. For more details, please see Tables 2 and   3.

Supplemental Note
To examine whether piR30840 has any homolog in mouse, we have also performed deep sequencing for small RNAs in the mouse CD4 T-lymphocytes. Unexpectedly, we found that the homogenous piRNA derived from U63 is lowly-expressed in the mouse CD4 lymphocytes (data not shown). Further sequence analysis suggested that the sequence of this piRNA was quite conserved in hominoidea, but not conserved between hominoidea and rodentia ( Figure S8A). In addition, the target binding site of human piR30840 is also not conserved in mouse ( Figure S8B). Although we tried to predict the binding between this piRNA and the second intron of mouse IL-4, the predicted binding site is significantly different from that of human and with poor stability ( Figure S8C).
Moreover, phylogenetic analysis and evolutionary distance estimation showed that the piRNA and its target region on IL-4 intron were subject to different evolutionary patterns, implying that this regulatory mechanism in human is not conserved in all mammals ( Figure S8D and E). By natural selection analysis, we found that the piRNA target region located on the second intron of IL-4 was under positive Darwinian selection, which may facilitate the specific match between the piRNA and its target loci on human IL-4 intron ( Figure S8F and G). To further confirm above conclusion, the experimental study was performed and result from western blotting implied that the extra three-nucleotide located in the target region on IL-4 intron of non-hominidae mammals obviously disturbed the effect of the piRNA on the expression of IL-4, strongly supporting the species specificity and the target specificity of this recent regulatory mechanism in evolution (about 13 million years ago, the divergence time between orangutan and human) ( Figure S8G and H)(1). Thus, these data suggest that the function of this piRNA is hominidae-specific, and the same regulatory mechanism does not exist in mouse.

Supplementary Figure 9. piR30840 induces the decay of IL-4 pre-mRNA.
(A) The effect of piR30840 on IL-4 pre-mRNA was examined in purified CD4 T lymphocytes from PBMCs. IL4 pre-mRNA was detected via qRT-PCR by using the primer pairs shown in Figure 4A.
(B) The construct pIL4 was co-transfected with s30840 or control into the 293T cells. IL4 pre-mRNA was detected via qRT-PCR by using the primer pairs shown in Figure 4A. Statistical significance between two samples was determined by using the student's t-test. P < 0.01. The data represents three independent experiments.
(C) Localization of PIWIL4 by using confocal microscopy. (C to E) The expression of TRAMP components such as trf4, air, and mtr4 were examined by using qRT-PCR.

Supplementary
(F to H) The expression of exosome components such as rrp42, rrp6, and rrp44 were detected by using qRT-PCR. Statistical significance between two samples was determined by using the student's t-test. P < 0.05.

Supplementary Figure 11. Ago protein interacts with IL-4 intron
(A to C) The tethering assay system was shown in Figure 4H (C) The copies of piR36170 (10 6 CD4 T lymphocytes) were detected by using qRT-PCR. The amount of loading was normalized with -actin mRNA.

SUPPLEMENTARY METHODS
Dual-luciferase assay. The reporter constructs containing the promoter, 3′UTR or 5′UTR of il-4 were co-transfected with the precursor or synthetic piR30840 using Lipofectamine 2000 (Invitrogen) into 293T cells.
The firefly and renilla luciferase activities were measured 48 hrs after transfection using a Dual-Luciferase reporter assay system (Promega). The firefly luminescence values were normalized to renilla luminescence values for transfection efficiency. N-Ago4 were generated via connecting the fragment of N with that of piwil4, ago3 or ago4 respectively.