Download The Role of the 3` UTR in Regulated Post

SHOWCASE ON RESEARCH
The Role of the 3' UTR in Regulated
Post-Transcriptional Gene Expression
Stan Stasinopoulos, Mythily Sachchithananthan and Robert Medcalf
Australian Centre for Blood Diseases, Monash University,
Alfred Medical Research and Education Precinct, VIC 3181
"...The end is important in all things" − Hagakure (The Way of the Samurai)
The control of protein production is essentially
achieved by regulating mRNA levels. Changes in
cytoplasmic mRNA steady state levels are a reflection
of the balance between the transcription rate and a
variety of post-transcriptional processes. Regulation of
these processes is conferred in part via a number of 3'
untranslated region (3' UTR) localised cis-acting
elements and their associated trans-acting factors. It is
therefore not surprising to find that mutations of
regulatory determinants in this region have been linked
to various pathological states including venous
thrombosis, neuroblastoma and myotonic dystrophy
(1). Following the initial transcriptional event, all premRNA must undergo a series of highly specific and
well orchestrated events to produce a mature
polyadenylated and translatable transcript. Essentially,
this sequence of events can be broken down into 5'
methyl capping, splicing, 3' end cleavage and
polyadenylation, nuclear-cytoplasmic transport and
translation. Following this, another series of events is
initiated to actively remove the transcript from the cell
(mRNA turnover).
The aim of this mini-review is to briefly outline the
mechanisms of regulated mRNA decay and 3' premRNA processing that we are investigating in the
laboratory to highlight some of the key events that
regulate gene expression.
circularised structure allowing the methyl-capped 5'
UTR to communicate with the 3'-polyadenylated tail
via the poly(A) binding protein/translation initiation
factor-eIF4G /methyl cap binding protein-eIF4Ebridging tertiary complex (2). Cytoplasmic mRNA
decay begins by exonucleolytic deadenylation via
poly(A) ribonuclease (Fig. 1). Subsequently the main
body of the deadenylated or oligoadenylated mRNA
is degraded in the 3'-5' direction by the exosome,
which is a multi-subunit complex composed of at least
10 proteins with 3'-5' exonuclease activity (3). There is
some emerging evidence in mammalian systems that
decapping precedes poly(A) shortening and that this
is a prelude for functionally relevant 5'-3' mRNA
decay (4,5). Nevertheless, at present 3'-5' mRNA decay
is considered to be the major mRNA decay pathway
in mammalian cells.
mRNA Stability
Regulation of mRNA decay rate (half-life) is a major
determinant of mRNA abundance in a cell. The half-life
of an individual mRNA can vary several orders of
magnitude, from minutes to days. Some of the most
highly regulated transcripts, under normal
physiological circumstances, are exquisitely unstable.
Often these transcripts encode members of the cytokine,
oncogene or transcription factor families, in effect,
proteins that by necessity are only transiently
expressed. Studies on these short-lived transcripts have
uncovered important information on posttranscriptional regulation and have also provided novel
approaches to manipulate gene expression at this level.
The regulated removal of mRNA from cells is a
complex process with a number of mRNA decay
pathways characterised to date, including the
deadenylation-dependent mRNA decay pathway,
endonucleolytic cleavage and the Nonsense-Mediated
Decay pathway. The principal means of mRNA
turnover is the deadenylation-dependant decay
pathway. Translationally competent transcripts adopt a
Vol 36 No 3 December 2005
Fig. 1. Schematic representation of various AU-rich
element (ARE) binding proteins interacting with an ARE.
(A) HuR can stabilise and potentially influence the
translation rate of a transcript.
(B) TTP may dislodge HuR and interact with the same
ARE element. In so doing, TTP can increase the rate
of deadenylation and the exosomal degradation of
the main body of the transcript.
AUSTRALIAN BIOCHEMIST
Page 13
3' UTR in Post-Transcriptional
Gene Expression
SHOWCASE ON
RESEARCH
Table 1. AU-rich elements (ARE) and their corresponding binding proteins (AUBP).
ARE
I
II
III
Class Sequence Characteristics
1-3 copies of a scattered pentamer, AUUUA, near or within a U-rich region
At least 2 copies of the nonamer, UUAUUUA(U/A)(U/A)
Non-AUUUA containing U-rich regions
AUBP
AUBP Characteristics
mRNA Target
Tristetraprolin
(TTP)
ARE mRNA destabilising protein that binds RNA via two CCCH zinc fingers.
Nuclear-cytoplasmic shuttling protein.
TNFα; GM-CSF; IL-2;
PAI-2
AUF1
Four isoforms via alternative splicing. Destabilises ARE mRNA. Remodels
ARE mRNA structure upon binding. Can stabilise ARE mRNA after heat shock.
c-myc; c-fos; groα; TNFα;
cyclin 1; GM-CSF
HuR/HuA
Ubiquitous ELAV-like ARE mRNA stabilising protein. Its activity is regulated
TNFα; u-PA; u-PAR;
by nuclear-cytoplasmic shuttling protein. Activity can be regulated by heat shock. VEGF; c-fos; p21
HuD; HuC; HuB Neuronal specific ELAV-like ARE dependant mRNA stabilising proteins.
GLUT1; MYCN; VEGF
NF90/NFAR1
IL-2
Double-stranded RNA binding protein that stabilises IL-2 mRNA.
Cis-Acting Elements and Trans-Acting
Factors Involved in mRNA Decay
Functionally relevant mRNA instability elements are
located throughout most unstable mammalian
transcripts, however they are predominantly found in
the 3' UTR. The best characterised of these are the
adenylate-and uridylate-rich (AU-rich) elements (ARE).
Indeed, bioinformatic studies indicate that
approximately 8% of human genes code for transcripts
that contain AREs (6). AREs differ in size and
sequence and have been classified into three general
classes (Table 1). The presence of an ARE can
modulate mRNA stability by varying the rate of
deadenylation, 3'-5' mRNA decay and even decapping.
The core ARE sequence is the pentameric motif
AUUUA, although often its capacity to destabilise a
transcript is greatly enhanced when it is located within
a longer nonameric motif (UUAUUUA[U/A][U/A]).
AREs exert their influence via specific interactions
with a variety of ARE binding proteins (AUBPs)
forming mRNA-protein (mRNPs) complexes. A
number of AUBPs have been characterised and shown
to influence mRNA decay rate in a regulated manner
(Table 1), and in some cases its translation and
subcellular localisation. For instance the AUBP
Tristetraprolin (TTP), can promote the decay of
transcripts including TNFα, IL-2, IL-3, GM-CSF, c-fos,
cox-2 and PAI-2, while the p37 and p40 isoforms of
AUFI can promote the decay of c-Myc (7). On the
other hand, the mRNA stabilising protein HuR can
stabilise the VEGF, GM-CSF, c-fos, p21, u-PA and uPAR transcripts (7).
The ultimate fate of an mRNA is determined by the
composition of an mRNP (i.e. protein-RNA and proteinprotein interactions) which, in turn, is regulated in a
sequence, cell-type and physiological state-dependent
manner. Despite intense studies over the last 10-15
years, it is still not clear as to how these AUBPs increase
Page 14
or decrease the decay rate of an mRNA. Certainly for
increased decay one can envisage a mechanism
whereby AUBPs can destabilise the circular nature of a
translationally competent mRNP thereby rendering the
mRNA susceptible to the cell's de-adenylation and
decay activities. TTP can increase both the rate of deadenylation of a transcript and the rate at which the
main body of the transcript is degraded by the
exosome. For decreased decay it is interesting to note
that TTP associates with the exosome, thereby
promoting the efficient assembly of the 3'-5' degradation
machinery on the mRNA in an ARE-dependant manner
(8) (Fig .1). A physiological role of these proteins
involved in mRNA turnover can be seen with TTP-/mice. These mice display an inflammatory phenotype
due to the excessive production of TNFα, the transcript
of which is normally turned over by TTP (9).
Regulation of mRNA Binding Proteins
The regulation of ARE-mRNA turnover is linked to
specific signal transduction pathways. The stability
and/or translation of a number of ARE-containing
transcripts is enhanced by the activation of the p38mitogen activated protein (MAPKAP) kinase 2 cascade.
Indeed, post-translational modification of AUBPs can
influence their ability to interact with an ARE element,
other proteins and their cellular localisation. For
instance, the AUF p37 and p40 isoforms are known to
act as ARE-dependent mRNA destabilising factors.
However, the phosphorylation status of the p40 isoform
can influence its capacity to destabilise mRNAs. Work
with THP-1 cells demonstrated that the IL-1β and
TNFα mRNAs were stabilised following treatment with
phorbol esters. This was accompanied by the reversible
loss of phosphate from the Ser83 and Ser87 residues of
the p40 isoform. The dephosphorylated p40 isoform
had a reduced ARE- binding affinity, but more
importantly, it altered the topology of the RNA to
AUSTRALIAN BIOCHEMIST
Vol 36 No 3 December 2005
SHOWCASE ON
RESEARCH
3' UTR in Post-Transcriptional
Gene Expression
which it bound, which in turn, altered the
architecture of the AUF1-ARE-ribonuclear protein
complex. It is possible that the altered architecture
either obscures the recruitment of factors that are
normally involved in ARE-mediated decay or,
conversely, it supports the binding of ARE-RNA
stabilising AUBPs (10, 11).
On the other hand TTP, apoptosis-promoting protein
TIA-1 and HuR are components of stress granules
which are formed during environmental stress and
result from the recruitment of translationally
incompetent initiation complexes. Here, these AREcontaining transcripts are either stabilised or
destabilised depending on the physiological state of
the cell. Under conditions of mitochondrial stress, TTP
is recruited to stress granules thereby increasing the
decay rate of ARE-containing transcripts. However
under different stress conditions, for instance arsenite
oxidative stress, TTP is not recruited to stress granules.
Under these circumstances TTP is phoshorylated by
MAPKAP kinase-2 at serines 52 and 178 creating a 143-3 binding site. This promotes the formation of a
TTP:14-3-3 complex which precludes TTP from being
recruited to stress granules and, consequently, stress
granule-localised ARE-containing transcripts are
shielded from decay (12, 13). Hence the cell has the
capacity to either increase or decrease TTP-dependent
mRNA decay depending on the stress conditions.
Emerging mRNA Decay Mechanisms
Recently a relatively new player in mammalian AREmediated decay was introduced by Yoshikuni
Nagamine and associates. They demonstrated that a
DExH-family RNA helicase, RHAU (RNA Helicase
Associated with AU-rich-element), was associated
with the ARE present in u-PA mRNA and played a
role in its rate of decay. A model was provided in
which RHAU recruits the RNA degradation
machinery containing the exosome and the poly(A)
ribonuclease to the ARE via its interaction with ARE-
binding proteins such as HuR and NFARI (14).
Potentially one of the most exciting advances in the
mRNA decay area was recently described by Jiahuai
Han and associates. In this groundbreaking work, the
authors demonstrated that ARE-mediated decay of
TNFα mRNA was dependent on ARE sequence
recognition by both TTP and the micro RNA, miR16.
TTP indirectly interacts with miR16 via its association
with the RISC (RNA Induced Silencing Complex)
protein Ago/eiF2C. They propose that the cooperation
between the two molecules, the microRNA, miR16,
and the AUBP, TTP, was essential for the efficient
recognition of the ARE sequence and subsequently for
ARE-mediated decay (15).
3' Pre-mRNA Processing
All mammalian pre-mRNAs undergo 3'endonucleoytic cleavage and virtually all mRNAs are
then polyadenylated, with the exception of histone
mRNAs in which the poly(A) tail is replaced by a
stem-loop structure (16, 17). The most important cisacting element for cleavage and polyadenylation is the
polyadenylation signal, AAUAAA, or its single
nucleotide variants (Fig. 2). The polyadenylation
signal is usually located 10-30 nucleotides upstream of
the cleavage site. A number of genes are known to
contain multiple polyadenylation signals. Hence, the
efficiency and accuracy of an individual 3' cleavage
site is determined by the polyadenylation signal itself
and the relative location of other cis-acting elements
upstream and downstream of the cleavage site.
Typically, in addition to the polyadenylation signal a
second cis-acting element, termed downstream
sequence element (DSE), usually located within 30
nucleotides of the cleavage is involved in defining the
site of cleavage. A consensus sequence has not
emerged for the DSE and it is loosely described as
GU/U-rich in sequence. The distance between the
polyadenylation signal and DSE determines the actual
site of cleavage.
Fig. 2. Schematic representation of the relative positions of the cis-acting elements involved in
mammalian cleavage and polyadenylation.
Represented are: the polyadenylation element (AAUAAA); the downstream sequence element (DSE);
the upstream sequence element (USE); the site of cleavage dinucleotide (CA).
Vol 36 No 3 December 2005
AUSTRALIAN BIOCHEMIST
Page 15
3' UTR in Post-Transcriptional
Gene Expression
SHOWCASE ON
RESEARCH
The two multisubunit proteins, cleavage and
polyadenylation specificity factor (CPSF) and cleavage
stimulation factor (CstF), interact with the
polyadenylation signal and DSE respectively. These
two proteins interact with each other and stablise the
binding of these complexes to the RNA with the aid of
cleavage factors (CF-1 and CF-2). The assembly of the
cleavage complex on the pre-mRNA directs the (as yet
unidentified) endonuclease to the site of cleavage.
There is no strict sequence requirement for cleavage,
however a CA dinucleotide is generally preferred.
Following the cleavage reaction, poly(A) polymerase
(PAP), which is recruited to the cleavage complex by
CPSF, catalyses the synthesis of the poly(A) tail. The
poly(A) tail is extended to approximately 200
nucleotides with the aid of poly(A) binding protein II
(PAB II), which interacts with the growing poly(A) tail
by successive binding.
A number of genes contain additional cis-acting
sequences besides the polyadenylation signal and DSE
that can influence cleavage site selection. One such
auxiliary element is located upstream of the
polyadenylation signal and is termed Upstream
Sequence Element (USE). USEs are loosely described as
U-rich in sequence and they appear to act as
recognition sites for trans-acting factors that aid in the
stable binding of mRNA 3'-end formation machinery
to the pre-mRNA (16).
Clearly, cleavage and polyadenylation is a highly
orchestrated mechanism, hence dysregulation of mRNA
3'-end formation can have drastic implications on gene
expression. The importance of cleavage and
polyadenylation in regulating gene expression is
demonstrated by the prothrombin G20210A
polymorphism, a G to A nucleotide substitution at the
cleavage site of prothrombin mRNA (18). This
polymorphism is associated with increased plasma levels
of prothrombin protein and an increased risk of venous
thrombosis (18). The mutant A residue at the cleavage
site is more efficiently cleaved and polyadenylated
compared to the wild type G allele (19-21). It would
appear that individuals carrying the A allele variant
produce increased amounts of prothrombin mRNA due
to increased efficiency in cleavage and polyadenylation.
This in turn increases prothrombin protein synthesis in
the plasma leading to an increased risk of developing
venous thrombosis. Further functional characterisation of
the various cis-acting elements involved in cleavage and
polyadenylation of prothrombin mRNA will enable a
greater insight into gene regulation of prothrombin at
this level.
Future Directions
mRNA decay is an important mechanism for rapidly
regulating gene expression in response to a signal
without the cell having to expend the vast amount of
energy that is associated with the transcription process.
Despite the current understanding of the enzymology
and mechanisms of ARE-mediated decay, a large
number of details still need to be resolved. For instance
we still need to determine how cis-acting elements, in
collaboration with various trans-acing factors,
Page 16
destabilise and even stabilise mRNA. As such, further
effort must be put into identifying and characterising
cis-acting elements and their trans-acting factors (i.e. the
mRNP) with respect to their interaction with the RNA
degradation machinery. The ultimate fate of a transcript
is influenced by the composition of an mRNP which, in
turn, is greatly influence by signal transduction
pathways. One envisages that investigating the
signalling pathways involved in this process will prove
to be a fruitful area for future research.
References
1. Conne, B., Stutz, A., and Vassalli, J. D. (2000) Nat.
Med 6, 637-641
2. Wells, S.E., Hillner, P.E., Vale, R.D., and Sachs, A.B.
(1998) Mol. Cell 2, 135-140
3. Butler, J.S. (2002) Trends Cell Biol. 12, 90-96
4. Cougot, N., Babajko, S., and Seraphin, B. (2004) J.
Cell Biol. 165, 31-40
5. Couttet, P., Fromont-Racine, M., Steel, D., Pictet, R.,
and Grange, T. (1997) Proc. Natl. Acad. Sci. USA 94,
5628-5633
6. Bakheet, T., Frevel, M., Williams, B.R., Greer, W.,
and Khabar, K.S. (2001) Nucleic Acids Res. 29, 246-254
7. Stasinopoulos, S.J., Tran, H., Chen, H.,
Sachchithananthan, M., Nagamine, Y. and Medcalf,
R.L. (2005) Progress in Nucleic Acids Research and
Molecular Biology 80, 169-215
8. Chen, C.Y., Gherzi, R., Ong, S.E., Chan, E.L.,
Raijmakers, R., Pruijn, G.J., Stoecklin, G., Moroni,
C., Mann, M., and Karin, M. (2001) Cell 107, 451-464
9. Carballo, E., Lai, W.S., and Blackshear, P.J. (1998)
Science 281, 1001-1005
10. Wilson, G.M., Lu, J., Sutphen, K., Sun, Y., Huynh, Y.,
and Brewer, G. (2003) J. Biol. Chem. 278, 33029-33038
11. Wilson, G.M., Lu, J., Sutphen, K., Suarez, Y., Sinha,
S., Brewer, B., Villanueva-Feliciano, E.C., Ysla,
R.M., Charles, S., and Brewer, G. (2003) J. Biol.
Chem. 278, 33039-33048
12. Stoecklin, G., Stubbs, T., Kedersha, N., Wax, S.,
Rigby, W.F., Blackwell, T.K., and Anderson, P.
(2004) EMBO J. 23, 1313-1324
13. Kedersha, N., and Anderson, P. (2002) Biochem. Soc.
Trans. 30, 963-969
14. Tran, H., Schilling, M., Wirbelauer, C., Hess, D.,
and Nagamine, Y. (2004) Mol. Cell 13, 101-111
15. Jing, Q., Huang, S., Guth, S., Zarubin, T.,
Motoyama, A., Chen, J., Di Padova, F., Lin, S.C.,
Gram, H., and Han, J. (2005) Cell 120, 623-634
16. Zhao, J., Hyman, L., and Moore, C. (1999) Microbiol.
Mol. Biol. Rev. 63, 405-445
17. Wahle, E., and Ruegsegger, U. (1999) FEMS
Microbiol. Rev. 23, 277-295
18. Poort, S.R., Rosendaal, F.R., Reitsma, P.H., and
Bertina, R.M. (1996) Blood 88, 3698-3703
19. Gehring, N.H., Frede, U., Neu-Yilik, G.,
Hundsdoerfer, P., Vetter, B., Hentze, M.W., and
Kulozik, A.E. (2001) Nat. Genet. 28, 389-392
20. Ceelie, H., Spaargaren-van Riel, C.C., Bertina, R.M.,
and Vos, H.L. (2003) J. Thromb. Haemost. 2, 119-127
21. Sachchithananthan, M., Stasinopoulos, S.J., Wilusz, J.,
and Medcalf, R.L. (2005) Nucleic Acids Res. 33, 1010-1020
AUSTRALIAN BIOCHEMIST
Vol 36 No 3 December 2005