The Xist RNA is a nontranslated transcript that is very large. It is important to understand the molecular structure of this important RNA. In the mouse a simple Northern experiment shows that the RNA is resolvable by agarose gel electrophoresis and would appear to be almost 18 kb

Molecular sizing experiments like those above indicated that the murine Xist RNA was larger than previously described. We have determined is that there is an additional exon/intron in the 3íend of the Xist gene.

 

Subsequently we determined that there was 3 kb of additional of sequence that was part of the Xist RNA.

These facts were demonstrated experimentally and are presented in the figure. We first determined that additional sequences were part of Xist, by querying NCBI and these results were confirmed experimentally. By analyzing the EST database at NCBI, we found four ESTs that mapped to the genomic region 3í to the Xist gene.

Subsequently, we were able to prove that all of these ESTs were found in Xist RNA from Female mice exclusively and finally that they could be found collinear on the same Xist RNA transcript. The lower part of the panel shows that there are a number of potential sites for polyadenylation in the 3íend of the Xist RNA. We wanted to know which if any of these sites were used in processing the RNA.

Northern analysis has shown that probe 850 detects the two transcripts. As there are two Xist RNA transcripts, we wished to determine what was different about them. The 854 probe only detects the longest Xist transcript. This would imply that the two transcripts are different at least in terms of the processing of their 3íend.

To evaluate which sequences at the 3íend of Xist play a significant role in polyadenylation we performed RNAíse protection experiments.
The probes in this figure span parts of Xist know to have candidate polyadenylation signals or where previous publications had claimed that the end of the Xist transcript could be found.

The results of the RNA protection experiments were quite convincing
The previously reported 3íend of Xist (probe P1) did not show a protection pattern consistent with a role as the true 3íend. In the new sequences that we had identified, two of the potential polyadenylation showed patterns of protection consistent with their role in the mature processing of the Xist transcript. Probe P2 protected two products, demonstrating that there were two disctinct transcript spanning this portion of the genomic DNA.

Finally probe P3 only protected a single product, demonstrating that the Xist RNA finally terminates in this region.