William Ou
55614127
November 24, 2015
News & Views
Just a month ago, a paper titled: An Xist-activating antisense RNA required
for X-chromosome inactivation, was published on Nature Communications. Contrast
to our understanding of X-inactivation via Xist thus far, these researchers found an
additional novel piece of long non-coding RNA expressed from the inactivated X
chromosome. They identified this lncRNA to be antisense of Xist, and that its
expression is required for proper Xist functioning. Here, I will explain the paper in 3
steps, 1) How they identified the novel antisense Xist RNA, 2) How they mapped it
and 3) How the function of the lncRNA is characterized.
First, as you can see from the diagram depicting the X inactivation center(XIC), Tsix
is a gene antisense of Xist, which have been shown from other literature and as
discussed in class, is a gene transcribed only from the active X chromosome in either
iXCI or random XCI. Since Tsix is also antisense to Xist, the researchers
distinguished the novel antisense lncRNA by breeding F1 rats with a mutant Tsix
allele (XΔTsix) such that the maternal X(Xm)is the containing the mutant allele. In
these rats, Tsix is expected to not be expressed, which they confirmed by RT-PCR
and sequencing RNA/DNA of male rats, since no inactivation is required in male
rats, Tsix is only expressed from that mutant allele (Figure 1b). However, in the
female versions of these rats, there appears to be an antisense transcript present,
which can only be transcribed from the WT inactivated Xp. This claim was
conclusively determined through sequencing by utilizing known SNPs in the
Xist/Tsix sequence. Next, to distinguish parent-of-origin-specific from strainspecific bias in expression, they compared RNAs produced from WT F1 hybrids and
their reciprocal counterpart. Through SNP profiling, they found Xist antisense
transcripts in both X chromosomes (active and inactive). In addition, they did a FISH
analysis, by probing the XistAR (probe shown in Figure 1a). Green is a probe for Xist,
red is for XistAR and white is for Atrx (transcribed from active X). Consistent with
speculations, XistAR probes coincided with Xist probes (as in figures). So far the
cells analyzed were from TS (trophoblast stem) and XEN (extra-embryonic
endoderm) which both exhibit iXCI. To examine the involvement of XistAR in
random inactivation, they examined antisense expression in EpiSC (epiblast stem
cells). Since it is impossible to deduce allele-specific expression in random
inactivation cell lines, they utilized F1 hybrids that are Tsix-heterozygous. In these
hybrids, X inactivation would be in favor the X-chromosome with mutant
Tsix(XΔTsix). Again, XistAR was detected only from inactivated X (XΔTsix). This was
further confirmed by FISH of WT hybrid EpiSCs(lower % because XistAR from
different chromosomes have different sequences and the probe only probes one of
them).
Now that the antisense Xist RNA is confirmed, the researchers set to map it out.
First, they used 5’ and 3’ RLM-RACE and found a 5’ cap on the RNA but failed to find
a poly-A tail on the 3’ end. The researchers also sequenced XistAR by RNA FISH (as
shown on the slide). Primers used that resulted in amplification, corresponds to the
sequence of the XistAR gene. The 5’ end mapped out to be bp 2802 of Xist and 3’ end
bp13 which is within exon 1 of Xist.
Lastly, the function of XistAR was characterized by insertion of intronic cassette
sequence containing multiple polyadenlyation (mpA) in the antisense orientation.
This intron insertion would lead to Xist RNA unharmed and XistAR truncated (XXpA).
William Ou
55614127
November 24, 2015
News & Views
As a control they analyzed a strain with the same intronic cassette but with no
mpA(XXIVS). As you can see on the graphs, the truncated XistAR resulted in decrease
in Xist expression. Since no Xist means no X-inactivation, ultimately means increase
expression of genes that are normally expressed from the active X. This is consistent
with data shown in Figure 6d. (RNF12, Atrx, Pdha1 are expressed from active X, utx
escapes inactivation shown by other papers).
The researchers had to determine the role of XistAR in both imprinted X-inactivation
and random X-inactivation. To do so they utilized a number of techniques. Select all that
apply.
1) In FISH analysis, the EpiSC cells showed a lower percentage of XistAR
associated with the silenced X chromosome (and Xist) compared to TS and XEN
cells because X-inactivation in EpiSC doesn’t normally happen
2) The researchers were able to utilize intronic cassettes to truncate XistAR and not
Xist because the double-stranded cassette has different splice sequences on
each strand.
3) A mouse heterozygous for a mutant Tsix was used to study random inactivation
because this mutant phenotype would knockout random inactivation
4) In this paper SNPs allowed researches to study the effects of single nucleotide
base pair changes on gene expression
5) Through RT-PCR analysis, the researches found another piece of Xist antisense
RNA and that it is the same size as Tsix.
Answer : False, True, false, false, false.