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Results: 7

1.
Figure 5

Figure 5. Extensive splicing of pri-miRNA transcripts. From: Dynamic microRNA gene transcription and processing during T cell development.

(A) Location of intron-spanning primer pairs within the Mir-29b-1/29a and Let7c-2/let7b genes. (B) RT-PCR was performed on RNA extracted from Drosha deficient CD4+ T cells. PCR products were cloned and sequenced to determine splice junctions. (C) Flanking RNA strands were quantified by quantitative RT-PCR in Drosha deficient, Dicer deficient and control CD4+ T cells.

Francis F. Kirigin, et al. J Immunol. ;188(7):3257-3267.
2.
Figure 7

Figure 7. Poor evolutionary conservation of miRNA genes. From: Dynamic microRNA gene transcription and processing during T cell development.

Shown is the alignment of H3K4me3 (marking the promoter) and RNA with mammalian sequence conservation for two miRNA genes. Analyses were performed on Drosha-deficient CD4+ T cells. The miRNA gene domains (pri-miRNA) are indicated by the arrows. Note that only the regions corresponding to the mature miRNAs, the promoter and a few other interspersed regions display sequence conversion.

Francis F. Kirigin, et al. J Immunol. ;188(7):3257-3267.
3.
Figure 3

Figure 3. Impact of altered seed sequence on predicted targets. From: Dynamic microRNA gene transcription and processing during T cell development.

(A) Shown are the miR-17 and miR-21 subspecies sequenced. The seed sequences are highlighted. (B) The miR-17+1 subspecies has the same seed sequence as miR-302a and miR-106a, whereas the miR-17+2 subspecies has the same seed sequence as miR-302c. Venn diagrams of targets predicted by Targetscan. Indicated are the numbers of targets that predicted for the annotated seed of (C) miR-17 or (D) miR-21, and the +1 and +2 shifted seed. Overlaps indicate identical targets predicted for more than one subspecies.

Francis F. Kirigin, et al. J Immunol. ;188(7):3257-3267.
4.
Figure 2

Figure 2. Altered miRNA precursor processing at DN3 to DP stages of T cell development. From: Dynamic microRNA gene transcription and processing during T cell development.

(A) Expression levels of miR-106b, 93 and 25, which are all derived from the same polycistron. Distribution of mature miRNA subspecies derived from the (B) pre-mir-17 and (C) pre-mir-21 precursors. The data indicates the contribution each subspecies makes to the total mature miR-17 or 21 pool at each stage of T cell development. MiRNAs derived from both 5p and 3p arms are shown.

Francis F. Kirigin, et al. J Immunol. ;188(7):3257-3267.
5.
Figure 1

Figure 1. Dynamic miRNA expression during T cell development. From: Dynamic microRNA gene transcription and processing during T cell development.

(A) The indicated populations corresponding to distinct stages of T cell development from LSKs through to mature CD4SP and CD8SP thymocytes were FACS-purified then analyzed for miRNA expression by miRNA-seq. As a comparison, Pro-B cells (related population) and MEFs (unrelated population) were also analyzed. Shown is a heatmap of all mature miRNA species sequenced at least once. The data has been clustered based on Euclidean distance. Comparisons of miRNA profiles between (B) CD4SP and CD8SP thymocytes; (C) DN1 and Pro-B; and (D) LSK and MEF. r = Peason’s correlation. Examples of (E) miRNAs expressed at constant levels; (F) hematopoietic progenitor-specific miRNAs; (G) miRNAs that are transiently upregulated during T cell development; and (H) miRNAs with bimodal expression patterns.

Francis F. Kirigin, et al. J Immunol. ;188(7):3257-3267.
6.
Figure 6

Figure 6. Identification of independent pri-miRNA transcriptional units embedded within protein-coding genes. From: Dynamic microRNA gene transcription and processing during T cell development.

(A) The Mir-21 gene overlaps with 3′UTR of the overlapping protein-coding Vmp1 gene. The pri-mir-21 transcript can be identified as an independent transcript by the presence of an additional H3K4me3 peak (marking a promoter) within intron 10, and RNA transcription through introns 10 and 11 of the host Vmp1 gene. Additional examples of miRNA genes overlapping with protein-coding genes include: (B) Mir-700, (C) Mir-15a/16-1, and (D) Mir-1891. (−) = Gene on the minus strand. (E) Confirmation that the pri-mir-21 transcript is transcribed independently from the overlapping protein-coding gene. Shown are the locations of the primers used to detect the pri-mir-21 transcript. One primer is located within intron 10 of Vmp1, and thus specific for pri-mir-21. (F) The pri-mir-21 transcript accumulates only in Drosha deficient CD4+ T cells, indicating that it is a bona fide pri-miRNA transcript. All analyses were performed on CD4+ T cells.

Francis F. Kirigin, et al. J Immunol. ;188(7):3257-3267.
7.
Figure 4

Figure 4. Mapping the structure of miRNA genes expressed in T cells. From: Dynamic microRNA gene transcription and processing during T cell development.

Drosha deficiency results in the accumulation of pri-miRNA transcripts derived from independent transcriptional units. Shown are the bicistronic (A) Mir-29b-1/29a and (B) Mir-181a-1/181b-1 genes in CD4+ T cells. PolyA RNAs (i.e. full-length and spliced RNAs) were isolated from MACS-purified CD4+ T cells obtained from DroshaF/F CD4-cre and wildtype mice. Libraries were then constructed and analyzed by NGS (RNA-seq). The RNA reads were then mapped to the mouse genome. (−) = Gene on the minus strand. Shown is the relative RNA expression in WT and Drosha deficient CD4+ T cells. Note that the neighboring protein-coding Mkln1 gene is not affected by Drosha deficiency. (B) RNA-seq combined with ChIP-seq for histone modification facilitates the mapping of actively transcribed miRNA genes. ChIPs for the histone marks H3K4me3 and H3K36me3 were performed on nucleosomes isolated from CD4+ T cells. Libraries were then constructed and analyzed by NGS (ChIPseq). The RNA or ChIP reads were then mapped to the mouse genome. A H3K4me3 peak marks a promoter, while H3K36me3 is broadly deposited over a gene body. The boundary of a transcriptional unit is defined by the interval in which both the pri-miRNA transcript (RNAs-seq) and H3K36me3 map to, and the presence of a H3K4me3 peak at the 5′ end. Shown are examples of small miRNA genes (C) Mir-17~92a-1 and (D) Mir-142; and large miRNA genes (E) Mir-30d/30b and (F) Let7c-2/let7b.

Francis F. Kirigin, et al. J Immunol. ;188(7):3257-3267.

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