Characterization and quantitation of differential Tsix transcripts: implications for Tsix function

Abstract

In dosage compensation of female mammals, the accumulation of Xist RNA initiates silencing of one X-chromosome. Xist action is repressed by the antisense gene, Tsix, whose full-length RNA product is complementary to Xist RNA in mice. While previous work showed that Tsix transcription blocks the accumulation of Xist RNA, it is still unclear whether this repression requires the antisense RNA product or whether the antisense transcriptional movement is sufficient. A better understanding of potential mechanisms requires elucidation of Tsix RNA structure and determination of Tsix RNA copy number relative to that of Xist RNA. Previous work indicated that at least some of murine Tsix is spliced and that human TSIX truncates within the 3' end of XIST. Here, further characterization and quantitation of murine Tsix RNA reveal three new findings: first, in undifferentiated embryonic stem cells, Tsix RNA is present at 10-100-fold molar excess over Xist RNA. Second, only 30-60% of Tsix RNA is spliced at known exon-intron junctions. The nearly equal abundance of spliced and unspliced species leaves open possible roles for both isoforms. Finally, Tsix is spliced heterogeneously at the 5' end and most detectable splice variants exhibit only a 1.9 kb region of complementarity between sense and antisense RNAs. Implications for Tsix's possible mechanisms of action are discussed.

Tsixdefective in splicing is competent to establishXistsilencing

“…Tsix transcripts are peculiar in that although a fraction of them remain unspliced, various patterns of splicing all remove most of the region complementary to Xist RNA from the 40-kb primary transcript, except for a 1.9-kb region present in the proximal end of Xist, to yield the major processed products of length 2.7 kb and 4.3 kb (Sado et al, 2001;Shibata and Lee, 2003). In addition, the distal part of the processed Tsix RNA thus produced covers the region up to 1.8-kb upstream of the Xist transcription start site, where no transcription has been detected in the sense orientation.…”

“…Tsix transcripts are subject to splicing and polyadenylation, like common protein-coding RNAs (Sado et al, 2001;Shibata and Lee, 2003). The major products of spliced Tsix RNA in undifferentiated ES cells, which are about 2.7 kb and 4.3 kb in length on northern blots, appear to be produced by differential usage of several polyA signals found in exon 4 of Tsix (Sado et al, 2001).…”

“…The major products of spliced Tsix RNA in undifferentiated ES cells, which are about 2.7 kb and 4.3 kb in length on northern blots, appear to be produced by differential usage of several polyA signals found in exon 4 of Tsix (Sado et al, 2001). RT-PCR-based methods also revealed the presence of some variants produced by alternative splicing (Sado et al, 2001;Shibata and Lee, 2003). In addition, Tsix RNA exists in spliced and unspliced forms, although the functional relevance of splicing remains unknown.…”

“…In addition, Tsix RNA exists in spliced and unspliced forms, although the functional relevance of splicing remains unknown. Because a significant fraction of the RNA is processed, and the splicing variants identified so far all share exon 4, which is the only exon possessing complementarity with the Xist sequence, it had been postulated that the splicing products (especially the sequence in exon 4) play an important role in the function of Tsix by masking the Arepeats in Xist, a region known to be required for Xist-mediated silencing (Wutz et al, 2002), through base pairing (Shibata and Lee, 2003).…”

Eukaryotic regulatory RNAs: an answer to the ‘genome complexity’ conundrum

“…Like Xist, Tsix lacks a conserved ORF and is found only in the nucleus . In undifferentiated female ES cells, Xist and Tsix are coexpressed on both X chromosomes, although the Tsix levels are in 10-to 100-fold molar excess over Xist RNA (Shibata and Lee 2003). However, a recent study suggests that Xist is expressed at an extremely low level prior to XCI and that Tsix is the major RNA component detected at the Xist/Tsix locus in undifferentiated ES cells (Sun et al 2006).…”