Download The indentification of protein-RNA interactions within the 5

120s Biochemical Society Transactions ( I99 I) I 9
The
indentification
of
protein-RNA
interactions within
the 5'untranslated
region of human preproinsulin mRNA.
STUART W . KNIGHT and KEVIN DOCHERTY
of
Department of Medicine, University
Birmingham,
Queen
Elizabeth Hospital,
Birmingham, B15 2TH, U.K.
The
biosynthesis
of
insulin
is
controlled at both transcriptional [l] and
translational levels [2]. A rise in the
plasma level of
glucose
triggers an
increase
in
both
de
novo
insulin
synthesis and secretion of stored insulin.
In
the
short
term the increase in
biosynthesis is actinomycin D resistant
implying the usage of pre-existing mRNA
[3]. There is a redistribution of alreadyinitiated preproinsulin
mRNA from the
cytoplasmic ribosome
fraction
to the
membrane-bound polysome fraction mediated
by the signal recognition particle [4] in
response to increasing glucose levels.
The specificity of the glucose-induced
increase
in
insulin
biosynthesis is
probably mediated via interactions with
the preproinsulin mRNA.
The 5'untranslated region (5'UTR) of
the human preproinsulin mRNA consists of
59
nucleotides
upstream
of the AUG
translation start codon [5]. There is a
high degree of sequence homology between
species in this region: the rat I and I1
5'UTRs have respectively 59.4% and 62.5%
homology with human mRNA whereas monkey
has 88.1% homology [6]. The region does
not have any palindromic sequences which
favour the creation of a hairpin loop
secondary structure [7] or an upstream ORF
181. Both of these features have been
implicated
in
the
regulation
of
translation.
We were interested in evaluating the
role of the human preproinsulin 5'UTR
mRNA sequences in the glucose-mediated
control of translation. To investigate
protein binding to the 5'UTR of the human
preproinsulin
mRNA
a
set
of
RNA
oligonucleotides
were
synthesised
corresponding to this region. The RNAprotein interactions were then analysed
using gel retardation.
Five overlapping RNA oligonucleotides
corresponding
to
the
5'UTR
were
synthesised from DNA oligo templates: A(-1
to -2O), B(-11 to -30), C(-21 to -40), D(31 to - 5 0 ) , and E(-41 to -59). These DNA
templates contained the 17 nucleotide T7
RNA polymerase primer recognition sequence
at
their
3'
end
facillitating the
transcription of the RNA oligos using TI
[9]. The transcription
RNA polymerase
reaction gave a mixture of RNA sequences
of varying length; the full length RNA was
purified using a denaturing acrylamide
gel. The RNA-protein interactions were
then assessed by gel retardation using a
native 6% polyacrylamide gel. For the
preparation of protein extracts cells were
lysed in ice cold 1% Triton X-100, 25mM
Tris-HCL (pH
7.5), 40mM
KC1 in the
presence of protease inhibitors, and then
centrifuged at 100,000 g for 60 minutes at
4" C to remove cell debris and membranes.
Protein extracts from both non-insulin
secreting cell lines (monkey kidney COS 7
and human liver HEP G2), and insulin
secreting cell lines (rat pancreatic pcell RIN m5f and the Syrian hamster HIT
T15 cell line) were utilised.
The COS I and HEP G2 extracts gave
banding patterns
of greater than two
retarded bands for each RNA sequence, RIN
m5F and HIT T15 extracts gave much less
well defined bands, but this may be due to
the presence of nuclear proteins or lower
abundance of the particular proteins in
these extracts. The sequence specificities
of these protein-RNA interactions were
determined using excess cold RNA sequence.
The interaction of protein(s) with
RNA
sequences A (-1 to -20) and E (-41 to -59)
were abolished by prior incubation with
excess
concentrations
of
all
other
unlabelled RNA sequences. RNA sequence B
(-11 to -30) and C (-21 to -40) were
specifically competed by excess A (-1 to20), B (-11 to -30) and C (-21 to -40).
The interaction of protein(s) with RNA
sequence C (-21 to -40) were competed with
excess C (-21 to -40) and D (-31 to -50),
while the proteins binding to RNA sequence
D (-31 to -50) were competed with excess C
(-21 to -40), D (-31 to -50) and E (-41 to
-59). These data indicate two sites of
specific protein interactions within the
human preproinsulin mRNA 5' UTR localised
in the regions (-11 to -30) and (-31 to50).
These
preliminary
studies
have
identified sequence-specific protein-RNA
interactions
within
the
human
preproinsulin mRNA 5'UTR. The identity of
the proteins and the significance of their
interaction remains to be elucidated. The
cell-type distribution
of
these RNAbinding proteins is not restricted to
insulin secreting cells. This lack of cell
sepecific expression
does not exclude
these proteins from a role in the cellular
mechanism of preproinsulin translational
control. Further information concerning
the molecular weight of the RNA-binding
proteins involved can be obtained by UVcrosslinking and SDS-PAGE analysis.
S.W.Knight is supported by a Medical
Research Council research studentship
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