The function of TIS11 family members was unknown at the time of their discovery and these early studies had implicated a role for TTP as a transcription factor. In a series of seminal experiments using inflammatory cells derived from TTP-deficient mice, it was discovered that TTP acts on a post-transcriptional level to promote rapid decay of ARE-containing mRNAs by directly binding to the ARE [48
]. A number of studies have determined that TIS11 members specifically bind the sequence UUAUUUAUU, which can be considered the core destabilizing element of many ARE-containing mRNAs [49
]. The binding of TIS11 members to ARE regions of mRNA transcripts depends on the integrity of the (Cys3
His) residues of TTP’s two zinc-finger domains [54
]. To this extent, a single mutation from cysteine to arginine in either of the zinc-finger domains attenuated ARE binding, and the non-binding mutant version of TTP exerted a dominant-negative effect over wild-type TTP with regard to promoting ARE-mediated decay [57
The initial work demonstrating the function of TTP as an mRNA decay factor arose from findings that showed TTP’s ability to inhibit TNF-α production in macrophages via
its binding to the ARE present in the TNF-α mRNA transcript [48
]. Since this initial discovery, TTP has been identified to bind the AREs present in many immune and inflammatory mediators and limit their expression via
ARE-mediated decay (). Furthermore, the role of TTP as a potential tumor suppressor is coming into light by virtue of its ability to mediate rapid decay of factors associated with tumorigenesis. More recent genome-wide assessment of TTP targets has identified novel mRNA targets and brought to light some of the complexities associated with TTP-mediated mRNA decay. In studies using TTP-deficient mouse embryo fibroblasts, over 250 mRNAs were stabilized in the absence of TTP, notably the ARE-containing gene Ier3 was identified and this finding points to a role for TTP in controlling blood pressure and cardiac hypertrophy [22
]. In similar microarray-based studies using macrophages, >100 mRNAs were identified to directly be associated with TTP and the 3′UTRs of these genes were enriched in ARE motifs, one in particular being interleukin-10 (IL-10) [25
]. An unexpected finding in this study demonstrated that in TTP-deficient macrophages the expression levels of some TTP-associated mRNAs were not increased, which may reflect the underlying redundancy among TIS11 family members in their ARE mRNA targets. Alternatively, it may imply that these mRNAs are resistant to TTP-mediated mRNA degradation through an unidentified mechanism.
A key step in the initiation of ARE-mediated decay lies within TTP’s capacity to promote deadenylation of ARE-containing transcripts. This was initially demonstrated through experiments showing the ability of TTP to cause a shortening of the poly(A) tail to the TNF-α and GM-CSF transcripts [56
]. Efficient TTP-mediated deadenylation is strongly dependent on the core binding sequence UUAUUUAUU and it being bound by the two zinc finger domains of TTP and BRF members [53
]. To this extent, three major deadenylation complexes have been reported in vertebrates, the Ccr4/Caf1/Not complex, the Pan2/Pan3 complex, and the poly A-specific ribonuclease (PARN) complex [59
]. Co-immunoprecipitation experiments have shown direct association of Ccr4 with TTP and BRF-1 which leads to decay of ARE-containing reporter mRNAs [62
], and depletion of Caf1 allows for stabilization of ARE-containing transcripts [59
]. Moreover, in vitro
studies have demonstrated that TIS11 family members can stimulate PARN activity to promote deadenylation of ARE-containing, polyadenylated mRNAs [63
]. Interestingly, the association between TTP and PARN appears to be indirect, since these two factors do not physically associate [62
], thus it is possible that other factors yet to be identified may facilitate bridging between TTP and PARN in order to activate deadenylation [63
The process of deadenylation leaves the mRNA body susceptible to rapid decay, and in mammalian cells TIS11 family members can participate in two primary decay pathways: 5′-3′ decay that occurs in processing (P)-bodies which are small cytoplasmic foci that contain many of the enzymes required for mRNA decay, and 3′-5′ decay is mediated through a complex of exonucleases known as the exosome [64
]. Which pathway TTP preferentially directs ARE-containing mRNAs into is not currently known, thus it is unclear what determines the prevailing decay pathway. One possibility is that 5′-3′ mRNA decay in P-bodies may prevail under conditions of cellular stress. Cells exposed to various stresses such as heat shock, oxidative stress, or glucose deprivation promote the assembly of stress granules which are small cytoplasmic foci that harbor translationally arrested mRNAs, stalled translation initiation factors, and the ARE-binding proteins TIA-1 and TIAR [66
]. Under conditions of stress, TTP and BRF-1 can be recruited to stress granules where they have been shown to target and sequester ARE-containing mRNAs and, as visualized using immunofluorescence microscopy, stress granules and P-bodies make frequent yet transient contacts [67
]. This evidence indicates the dynamic relationship between stress granules and P-bodies to be dependant upon TIS11 family members where they may facilitate delivery of selected mRNAs from stress granules to P-bodies for degradation.
Another feature contributing to TIS11 family members directing ARE-containing mRNA to P-bodies is the ability of TTP and BRF-1 proteins to bind and deliver ARE-containing mRNAs to P-bodies [69
]. In this complex, TTP associates with P-body components involved with decapping such as Dcp1a, Dcp2, and Hedls, along with the 5′-3′ exonuclease Xrn1 to promote ARE-containing mRNA decapping and exonucleolytic degradation in a 5′-3′ direction [47
]. Additionally, central components of the RNA-induced silencing complex, Argonaute proteins, primarily reside in P-bodies [72
] and current work has implicated TTP as a novel mediator of microRNA-dependent mRNA decay and translational repression. Through its interaction with Ago2 and Ago4, TTP has been shown to facilitate targeting of an ARE-specific microRNA (miR16) to the TNF-α ARE [73
]. Although TTP does not directly bind miR16, they interact through association with Argonaute proteins to mediate ARE-containing mRNA silencing.
A main component involved in 3′-5′ mRNA degradation is the exosome. This multiprotein complex of exonucleases and RNA binding proteins is required for 3′-5′ decay [74
]. TTP and BRF-1 have been shown to interact with two components of the exosome PM-scl75 and Rrp4 [62
]. Although the exosome is not localized to P-bodies, these findings indicate that TIS11 family members can act outside cellular RNA decay centers and provide a functional link recruiting the exosome to ARE-containing mRNAs. Taken together, the TIS11 family member’s ability to regulate gene expression by targeting ARE-containing mRNAs for degradation is a complex interplay of various decay enzymes comprising a number of decay pathways that are functionally housed in unique cellular compartments. The fact that TTP and BRF proteins serve as a molecular link between ARE-containing mRNAs and these pathways indicates their significance in post-transcriptional gene regulation ().
TIS11 plays a central role in post-transcriptional regulation of gene expression