BLGY1232 RNA processing
Transcripts of eukaryotic protein-encoding genes are not usually immediately ready
for transport to the cytosol They must first be processed
Most transcripts are generated first as primary transcripts that undergo subsequent
processing The evidence for this comes from experiments in which cells in culture
were pulse-labelled with radioactive precursors of RNA
HnRNA: ‘Heterogeneous Nuclear’ RNA
Experiment:
1. Grow mammalian cells in culture
2. Incubate Hela cells (human cancer cell line) in culture with 32PO4 for a short
time-period (about 10 minutes) This will be incorporated into any RNA
made during this time
3. Transfer the cells to fresh medium containing unlabelled precursor (non-
radioactive PO4), and continue the incubation This “chases out” the
labelled precursor, so that further newly synthesised RNA is not labelled; This
is called a pulse-chase experiment
4. Harvest cells at intervals, extract RNA and analyse by gel electrophoresis
5. Autoradiography of the gel shows the fate of RNA labelled during the pulse
period
Results:
First, look at the pattern in the gel of the RNA bands stained with Ethidium
bromide - This stain will stain all the RNA in the cell and the stained RNA
comprises the steady-state content of the cell - all the bulk RNA that was
present in the cell, before, during and after the labelling period
We see 4 prominent bands, which correspond to the most abundant RNA
species in the cell: 26-28S rRNA, 18S rRNA, 5.8S rRNA and 5S rRNA Together,
these comprise 95%of the RNA in all cells (We may also see a very low
molecular weight band corresponding to tRNA, if it hasn’t run off the end of
the gel) mRNA is usually not visible as a stained band, because it consists
of very many different molecules of different lengths: it is present as a faint
background “smear”, which is not usually clearly seen.
Individual genes are simultaneously transcribed by multiple polymersases, to
generate multiple transcripts
At the end of the labelling period, we see:
(i) A single high-molecular weight labelled band corresponding to 45S RNA
(ii) A heavily labelled, heterogeneous “smear” of labelled RNA
(iii) A single heavily labelled 5S rRNA band
At subsequent timepoints, two things happen;
(i) The heavily-labelled 45S band disappears, and is replaced by 4 bands
corresponding to the rRNA components.
(ii) The “smear” becomes generally smaller in its average size-range, and also
becomes slightly less heavily labelled
Note: Cell fractionation experiments show that the high-molecular weight 45S and the
HMW “smear” are found only in the nucleus. The lower molecular weight products are
found in the cytoplasm. The HMW smear is sometimes referred to as “heterogeneous
Transcripts of eukaryotic protein-encoding genes are not usually immediately ready
for transport to the cytosol They must first be processed
Most transcripts are generated first as primary transcripts that undergo subsequent
processing The evidence for this comes from experiments in which cells in culture
were pulse-labelled with radioactive precursors of RNA
HnRNA: ‘Heterogeneous Nuclear’ RNA
Experiment:
1. Grow mammalian cells in culture
2. Incubate Hela cells (human cancer cell line) in culture with 32PO4 for a short
time-period (about 10 minutes) This will be incorporated into any RNA
made during this time
3. Transfer the cells to fresh medium containing unlabelled precursor (non-
radioactive PO4), and continue the incubation This “chases out” the
labelled precursor, so that further newly synthesised RNA is not labelled; This
is called a pulse-chase experiment
4. Harvest cells at intervals, extract RNA and analyse by gel electrophoresis
5. Autoradiography of the gel shows the fate of RNA labelled during the pulse
period
Results:
First, look at the pattern in the gel of the RNA bands stained with Ethidium
bromide - This stain will stain all the RNA in the cell and the stained RNA
comprises the steady-state content of the cell - all the bulk RNA that was
present in the cell, before, during and after the labelling period
We see 4 prominent bands, which correspond to the most abundant RNA
species in the cell: 26-28S rRNA, 18S rRNA, 5.8S rRNA and 5S rRNA Together,
these comprise 95%of the RNA in all cells (We may also see a very low
molecular weight band corresponding to tRNA, if it hasn’t run off the end of
the gel) mRNA is usually not visible as a stained band, because it consists
of very many different molecules of different lengths: it is present as a faint
background “smear”, which is not usually clearly seen.
Individual genes are simultaneously transcribed by multiple polymersases, to
generate multiple transcripts
At the end of the labelling period, we see:
(i) A single high-molecular weight labelled band corresponding to 45S RNA
(ii) A heavily labelled, heterogeneous “smear” of labelled RNA
(iii) A single heavily labelled 5S rRNA band
At subsequent timepoints, two things happen;
(i) The heavily-labelled 45S band disappears, and is replaced by 4 bands
corresponding to the rRNA components.
(ii) The “smear” becomes generally smaller in its average size-range, and also
becomes slightly less heavily labelled
Note: Cell fractionation experiments show that the high-molecular weight 45S and the
HMW “smear” are found only in the nucleus. The lower molecular weight products are
found in the cytoplasm. The HMW smear is sometimes referred to as “heterogeneous
