I Key : Primer direction
Exon 2-derived transcripts (any start site)
Exon 1 Exon 3 Exon 4
Nla IV 573 bases 530 bases 428 bases 260 bases 197 bases 168 bases
Key : - Transcription start sites
Figure 3 .1 7 -A diagrammatic representation o f the riboprobe used in the protection assay and the predicted fragments. RNase protection assays involve the annealing o f an antisense radiolabelled riboprobe (the arrow) to transcripts. Addition o f RNase results in the digestion o f any single-stranded RNA. Multiple transcription start site usage, as depicted above will lead to the production o f a multiplicity o f truncated riboprobes, which may be separated by PAGE and visualised by autoradiography. The expected fragment sizes using the Sau 3A-Nla IV riboprobe are indicated to the right.
Start site 1 Start site 2
v-^
573 bases 530 bases
Start site 3 428 bases
Deletion 260 bases
Start site 4 197 bases
Exon 2 168 bases
Figure 3.18 - Autoradiograph obtained fro m an RN ase protection assay using liver RN A ,
using the method described by Lowe et al. (1987). The riboprobe was incubated in the absence o f RNA, ethanol precipitated and loaded on the gel in the standard manner, as shown in the left-hand lane, marked probe. Riboprobe was also incubated with tRNA (marked tRNA) and RNased in the normal fashion, in order to confirm that the bands observed in the RNase protection assay o f liver RNA (marked liver RNA) were not due to incomplete digestion o f the riboprobe. Exon 1 transcription start sites I to 4 are marked on the left, along with those initiated from start sites 1 and 2 from which the 186 base deletion has been excised (deletion). Exon 2 has at least 2 start sites, but cannot be differentiated due to the probe used. A lengthy exposure was required in order to visualise transcription start site I within exon I. The sizes o f the fragments were ascertained from comparison with end-radiolabelled marker DNA and from the literature, and are given in bases to the right. The probe and tRNA lanes were
Protection assays on liver RNA extracted from 50-day old rats clearly show the bands
produced (Figure 3.18). The top band at 530 bases represents transcripts initiated at
start site 2 (ss2). The next two bands at 428 and 260 bases depict start site 3-initiated
transcripts (ss3) and those from start sites 1 and 2 from which the 186 base region has
been spliced (del), respectively. The band below these two, at 197 bases is produced by
transcripts initiated from start site 4 (ss4), whereas the smallest band at 168 bases
depicts exon 2-derived transcripts. Transcripts derived from start site 1 (ssl), at 573
bases in length, could only be visualised after much longer exposure.
Interestingly, no band was identified from RNase protection assays which would
correspond to a transcript from which 60-80 bases had been deleted, as suggested from
the RT-PCR amplifications o f whole liver, bone and osteoblast RNA, confirming that
this was an artefact (see section 1).
ii) Confirmation o f the Identity o f RNase Protection Assay Fragments
The identity o f the protected bands was confirmed by performing a protection assay on
six aliquots of liver RNA (40 pg each), and eluting the fragments from the gel
individually. These were amplified by RT-PCR using the appropriate primers specific
for each start site (see Appendix III). The RT-PCR products were ligated into a TA
vector and sequenced using flanking primers (see Appendix I for methodology). Three
examples o f sequence obtained are given in Figures 3.19-3.21. Comparison of the
sequence obtained with that previously published (Appendix III) revealed that the bands
Figure 3.19 - Sequence analysis to confirm the identity o f the band corresponding to exon I-derived transcripts initiated from start site 1.
Figure 3.20 - Confirmation o f the identity o f the RNase protection assay band representing transcripts initiated from start site 2 o f exon 1.
CD O o o o o CD C u CD CD C = C< < CD CD CD CD C CD % Ü O z c CD - C CD CD 1 8 0
Figure 3.21 - Characterisation of the band, obtained from RNase protection assays, representing exon 1-derived transcripts initiated from start site 3.
3
’i X Cl < J <ni) RNase Protection Assay Optimisation
In my hands, the RNase protection assay procedure described by Lowe et al (1987)
required surprisingly lengthy exposure before any bands could be visualised. For
instance, IGF-I mRNA levels are known to be high in liver; however, bands could only
be detected after exposure o f between 24 to 48 hours for 20pg o f RNA. Osteoblasts
and bone were unlikely to contain a similar abundance o f IGF-I mRNA and in order to
minimise exposure-time, the conditions were optimised. This was undertaken as
suggested by Lau et al (1992), using a range o f RNase A:Ti concentrations between
40:2pg/ml to 10:0.5pg/ml respectively. Simultaneously, the incubation time was also
varied from between 15 to 60 minutes.
The effects o f these changes can be clearly seen in Figure 3.22. This figure shows an
example o f an autoradiograph obtained after 15, 45 and 60 minutes o f digestion. The
concentration o f RNase A:Ti used in each panel was increased from 10:0.5pg/ml (used
by Lau et al., 1992) to 40:2pg/ml as used by Lowe et al (1987).
The bands obtained after 15 minutes incubation, using the minimum amount o f RNase,
were most intense, and could be easily visualised after 24 hours exposure. However,
using increasing concentrations of RNase led to a rapid reduction in signal. For
example, when the highest concentration o f RNase was used, the autoradiograph had to
be exposed to film for at least four days to obtain a measurable signal, demonstrating
that while the protected fragments were present, they were less abundant. In contrast,
the duration o f the digestion appeared to play a minor role. This resulted in a slight
reduction o f band intensity from that observed for each RNase concentration after 15
Digestion Time 15 min
h
4 RNase T, 10 20 30 40 RNase A 0.5 1.0 1.5 2.0 ss 2 ss 3 Del ss 4 Ex 2* é
# # #
45 minh
H 10 20 30 40 0.5 1.0 1.5 2.0 60 minh
10 20 30 40 0.5 1.0 1.5 2.0 iFigure 3.22 - RN ase protection assay optimisation. The RNase digestion protocol was optimised
fo r time, from 15 to 60 minutes, and RNase concentration. The RNase concentrations used varied between RNase A: RNase T, 10 pg/ml:0.5 pg/ml to 40 jug/ml:2 pg/ml respectively. The optimisation assays were performed in parallel and the autoradiographs were exposed fo r the same length o f time. It is clear that while the incubation time affects the intensity o f the signal produced, the concentration o f RNase used plays a more critical role. The conditions given by Lowe et al. (1987) involved the use o f the highest concentration o f RNase fo r 60 minutes, under which no bands are visible except after lengthy exposure. The start sites are denoted as s s l -4. Del refers to those transcripts from which the 186 base region has been alternatively spliced and exon 2-derived transcripts are denoted Ex 2. The protection assays shown in the three panels were exposed fo r 24 hours.
Exon 2 Signal