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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. 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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