Biology Reference
In-Depth Information
before transcription of pre-mRNA is even complete, there is very little outron-contain-
ing pre-mRNA available from which a transcriptional start site can be identified.
Recent experiments have shown that nested primers can be used to determine
outron lengths and approximate transcriptional start sites by RT-PCR (
Morton and
Blumenthal, 2011
). In addition, when chromatin immunoprecipitation (ChIP) with
an antibody recognizing RNA polymerase II was performed on starved C. elegans
larvae, deep sequencing of the precipitated DNA products revealed accumulated
polymerase upstream of many genes, possibly paused at promoters (
Baugh et al.,
2009
). Likewise, C. elegans ChIP with an antibody recognizing the histone variant
H2A.Z (HTZ-1), followed by hybridization to DNA microarrays, indicated that this
type of histone is also often found just upstream of genes, possibly marking active
promoters (
Whittle et al., 2008
). The genomic sites of these polymerase and HTZ-1
peaks correspond closely to the putative start sites of several outrons analyzed by RT-
PCR (
Table I
). Finally, minimum outron lengths can be estimated from occasional
ESTs representing untrans-spliced RNA. Data from these multiple measures of
outron length indicate that average outrons are around 300 nt, although examples
of shorter and significantly longer outrons have also been observed (
Morton and
Blumenthal, 2011
).
5. Additional Components of the SL1 snRNP
At the conclusion of the trans-splicing reaction, the outron becomes attached to
the 78 nt 3
0
portion of the SL snRNP as a Y-branched molecule, analogous to the
lariat byproduct associated with intron splicing (
Bektesh et al., 1988
). This molecule
is subsequently debranched and degraded, although the Sm proteins associated with
this branched molecule may be recycled. The fate of the components of the truncated
snRNP is an interesting question because, unlike the U snRNPs used in cis-splicing,
the SL snRNPs required for trans-splicing are consumed in each reaction.
Table I
Estimated outron lengths
Gene
Outron (RT-PCR)
Longest EST
ChIP-seq
rsp-3
247-255
All trans-spliced
250
rps-3
288-310
All trans-spliced
310
vha-6
363-506
All trans-spliced
No peak
Y37E3.8
263-321
All trans-spliced
270
col-13
57-100
65
85
pas-3
82-146
164
No peak
idh-2
302-347
304
310
Note: These genes have all been analyzed by RT-PCR, using forward primers in the outron located successively upstream
of the gene
'
s trans-splice site. The transcriptional start site was determined to be in the region between the last forward
primer from which a product could be obtained and the next forward primer upstream of this site. ESTs were taken from
Wormbase, WS210. ChIP-seq data is from Baugh et al. (2009), analyzed after being loaded as a custom track on the
UCSC genome browser (
http://genome.ucsc.edu/cgi-bin/hgGateway
). A peak of polymerase was not identified upstream
of all genes.
Search WWH ::
Custom Search