Abstract
Eukaryotic gene expression is mainly controlled at translational level. Compared with
transcriptional regulation it allows for more rapid changes in cellular concentrations
of proteins in response to stress events. The most regulated phase of translation is an initiation
step. The initiation occurs by scanning of the 5′-terminal regions of mRNAs by the initiation
complex or according to the cap-independent mechanism. The 5′-terminal regions of mRNAs
modulate translation efficiency and together with protein factors participate in translation
regulation. It has been observed that the 5′-terminal region of p53 mRNA is involved
in regulation of expression of p53 protein and its major isoform Np53 at translational level.
This region may exist in several different sequence variants due to the presence of multiple
transcription start sites, alternatively spliced intron sequences and alternative translation
initiation codons. It has also been suggested that the 5′-terminal region of p53 mRNA exerts
properties of an IRES element, which enables internal initiation of translation.
In this dissertation the impact of different variants of the 5′-terminal region of p53
mRNA on translation of reporter protein has been investigated. The mechanism of translation
initiation from AUG1 start codon for full-length p53 and AUG2 for Np53 isoform has also
been evaluated. The variants of the 5′-terminal region of p53 mRNA were followed
by the luciferase coding sequence in model mRNA constructs. They were translated in vitro
in rabbit reticulocyte lysate, as well as in HeLa and MCF-7 cells. Structural probing of the
5′-terminal region of p53 mRNA in the model mRNA construct confirmed that AUG1 codon
is embedded within stable hairpin G56-C169. This hairpin might cause a steric hindrance
for the moving ribosome, thus increasing translation efficiency from the AUG1 codon.
This hypothesis was supported by toe-printing analysis, in which initiation complexes
at AUG1 codon were hardly detected. The results of in vitro translation studies suggested
that
the
translation
initiation
from
AUG1
codon
was
mostly
cap-dependent.
However, translation initiation from AUG2 showed rather IRES-dependent character.
It turned out that the region spanning two transcription start sites P0 and P1 strongly reduces
the translation level from AUG1 codon. It seems to be a consequence of stable secondary
structure of the cap-proximal mRNA region, which may interfere with scanning
by the ribosome. In another mRNA construct, the presence of intron 2 resulted in translation
initiation only from AUG2 codon. Moreover, mutation of UGA STOP codon present within
intron 2 to GCG revealed that AUG1 codon is also active in translation. Thus, uORF
is
formed
in
this
mRNA
variant,
which
encodes
25-aminoacid-long
peptide.
Therefore, Np53 isoform might also be generated from not completely spliced p53 mRNA
and its synthesis occurs according to reinitiation model after synthesis of a short peptide from
AUG1 codon.
In order to modulate translation efficiency of p53 protein and Np53 isoform,
antisense oligonucleotides complementary to the 5′-terminal region of p53 mRNA were
applied. These oligomers, which were able to bind to the most characteristic structural
elements of 5′-terminal region, hairpin G56-C169 and U180-A218, induced changes
in translation level from both the initiation codons during translation in rabbit reticulocyte
lysate. This indicates, that both hairpins play a crucial role in translation initiation.
Moreover, it turned out that the remaining RNase H activity in the lysate contributed
to the effect of antisense oligomers on translation efficiency. The 2′-O-methylated
and LNA-gamper derivatives of selected oligomers were also be used to reduced p53 protein
level in MCF-7 cells. These oligomers are good candidates to be applied in the future
to reduce p53 level in radio-resistant tumor cells.