Identification of a novel circularized transcript of the AML1 gene - PubMed (original) (raw)

Identification of a novel circularized transcript of the AML1 gene

Ai-ning Xu et al. BMB Rep. 2013 Mar.

Abstract

The AML1 gene is an essential transcription factor regulating the differentiation of hematopoietic stem cells into mature blood cells. Though at least 12 different alternatively spliced AML1 mRNAs are generated, three splice variants (AML1a, AML1b and AML1c) have been characterized. Here, using the reverse transcription-polymerase chain reaction with outward-facing primers, we identified a novel non-polyadenylated transcript from the AML1 gene, with exons 5 and 6 scrambled. The novel transcript resisted RNase R digestion, indicating it is a circular RNA structure that may originate from products of mRNA alternative splicing. The expression of the novel transcript in different cells or cell lines of human and a number of other species matched those of the canonical transcripts. The discovery provides additional evidence that circular RNA could stably exist in vivo in human, and may also help to understand the mechanism of the regulation of the AML1 gene transcription.

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Figures

Fig. 1.

Fig. 1.. Analysis of RT-PCR results. (A) Detection of the novel AML1 transcript with the exon 5-6-5 alignment using RT-PCR and sequence analysis. Lanes 1 and 4 show the amplification products of the novel transcript in an AML patient and a healthy individual respectively, Lanes 2 and 5 are negative control, and Lanes 3 and 6 are blank control. The band above 305 bp in Lane 4 is nonspecific amplification product. The positions and directions of outward-facing primers (oF and oR) in the cDNA structure are indicated by arrows. The sizes of the expected fragment and the junction sequence of the 3’ end of exon 6 and 5’ end of exon 5 are also shown. (B) Detection of exon 5-6-5 alignment using different pairs of primers. Positions and directions of different pairs in the cDNA structure are indicated by arrows. The amplification products of primers 6F/5R2, 5F2/5R1, F65/5R3 and 5F1/R65 are shown. By sequencing, the bands of 118 bp and 284 bp in Lanes 1 and 4 and the bands of 116 bp and 315 bp in Lanes 7 and 10 respectively are the products, Lanes 2, 5, 8 and 11 are negative controls, Lanes 3, 6, 9 and 12 are blank controls. (C) Analysis of RT-PCR results and schematic diagram of the positions and directions of primers used to seek the neighboring exons of the exon 5-6-5 alignment. Lanes 1 to 4 are products of RT-PCR using 3F/7aR, 3F/7bR, 4F/7aR and 4F/7bR, respectively. Asterisks show the canonical transcripts without exon 6. Lanes 5 to 8 are products of 3F/R65, 4F/R65, F65/7aR and F65/7bR, respectively. Lane 9 shows the amplification product of F65/6R, Lane 10 is negative control.

Fig. 2.

Fig. 2.. Observation of poly(A) tail in the novel transcript and expression of the novel transcript in human normal cells, cancer cell lines and several other species. (A) RT-PCR results. 1 μg total RNA was subjected to reverse transcription using either random primers or oligo(dT) primer and amplified by PCR using three pairs of primers oF/oR, F65/6R, and 5F1/R65. The canonical transcript was amplified using 4F/5R1. (B) RT-PCR results of total RNA, poly(A)+ RNA and poly(A)- RNA. Canonical transcripts were amplified using primers 4F/5R1 (shown on the left). Results of PCR amplifying the novel transcript are shown on the right. (C) Lines 1 to 2 respectively represent the amplification product of Oral mucosa and nucleus pulposus cells of an intervertebral disc using primers 5F1/R65; Lines 3 to 7 respectively represent U266, K562, SMMC-7721, AGS and rat NRK52E cells; Line 8 represents tetrahymena; Lines 9 to 10 respectively represent Euplotes elegans and Tartary buckwheat. The positive control represents cDNA from bone marrow of healthy individuals, followed by the negative control.

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