1. The role of PABP in translation initiation
All eukaryotic mRNAs have a cap structure at the 5′ end. As early as 1976, Shtkin pointed out based on the results of in vitro translation experiments, The 5′ end cap can enhance translation efficiency. Since then, many studies have confirmed that the translation of most mRNAs depends on the cap structure.
In addition to the cap, most eukaryotic mRNAs have a polyA tail at the 3' end. It has been observed in many in vivo experiments and highly active in vitro translation systems that the mRNA polyA structure is directly related to translation efficiency. , the mRNA with polyA is translated much more efficiently than the corresponding mRNA without polyA tail. The 5′-end cap and 3′-end polyA can coordinately regulate the translation efficiency of mRNA. Further studies have shown that during the initiation of eukaryotic translation, polyA is bound by PABP and affects translation through PABP.
PABP is highly conserved in eukaryotes and contains 4 RNA recognition motifs (RNA recognition motifs, RRM). Sachs et al. first demonstrated that PABP is involved in translation initiation. PABP can assist the combination of 60S subunit and 40S subunit to promote the formation of 80S ribosome [5]. Biochemical evidence also reveals the role of PABP in translation initiation. Neither polyA nor the 5'-end cap structure can act alone on translation, but can only act synergistically. PABP participates in the interaction between the cap and its initiation factor in this process [3, 6]. It is possible that PABP can interact directly with CBP or indirectly through an intermediary (as shown in Figure 1). Through this interaction, the two ends of the mRNA are very close in space to form a loop. This is consistent with the experimental results of electron microscopy observed more than 40 years ago that polyribosomes are ring-shaped. Perhaps eukaryotes improve translation efficiency through the interaction of both ends.
Figure 1 Interaction between the two ends of translation-initiating mRNA
If exogenous polyA is added to the in vitro translation system of lysozyme, protein synthesis is inhibited, which indicates that exogenous polyA binds (squester) a component necessary for translation. Gallie et al. also found that the inhibitory effect of mRNA without a cap structure was greater than that of mRNA with a cap, indicating that mRNA with a 5' end cap can efficiently compete for a component that is easily bound by exogenous polyA. Moreover, the addition of purified eIF4F and eIF4B reversed the inhibitory effect caused by polyA. It can be seen that the components bound by this exogenous polyA are eIF4F and eIF4B. Although these factors can directly act on polyA, their affinity to polyA is only about one-half of their affinity to PABP [7]. The most likely explanation for this is that the binding of polyA to eIF4F and eIF4B is completed through the protein-protein interaction between the PABP/polyA complex and each factor.
In yeast and plants, PABP directly interacts with the large subunit eIF4G (eIFiso4G) of eIF4F (eIFiso4F) to promote the binding of the 40S subunit to mRNA [7, 8]. However, mammalian PABP does not interact directly with eIF4G. Recently, a PABP-acting protein, PAIP-1, with certain homology to eIF4G was discovered in mammals. Craig et al. [9] proposed a model in this regard, believing that mammalian PABP and eIF4A use PAIP-1 as an intermediary to form a bridge between polyA and 5'-UTR. The synergistic effect of the 5' end cap and polyA on translation initiation may be It is completed according to the following steps: eIF4A is recruited to the 5′-end cap by interacting with eIF4G, and the cap in turn promotes the recruitment reaction of eIF4A (Figure 1), and then eIF4A interacts with PABP through PAIP-1 as an intermediary [4, 9 〕. In plants, not only eIF4F (eIFiso4F) and eIF4B can respectively increase the affinity of PABP for polyA, but they can also synergistically affect the binding ability of PABP to polyA. It is suggested that there must be a functional interaction between PABP, eIF4F and eIF4B [7].
In mammals, the content of eIF4F is low. In order to improve translation efficiency, eIF4F is combined with PABP to increase their binding to cap and polyA respectively [4].
The molecular interaction between PABP and its related initiation factors is controlled by the concentration of PABP and mRNA between cells. At a certain concentration, polyA (probably acting together with PABP***) can increase selectivity Translation of mRNA in vitro. Moreover, the intermolecular interaction involving PABP at both ends plays a role in detecting the integrity of pre-translational mRNA, thereby preventing the translation of incomplete mRNA. Another reason why PABP participates in intermolecular interactions in initiation may be to promote reinitiation by bringing the two ends closer together. Evidence has shown that the 40S subunit is still bound to the mRNA after translation; ribosomes bound to the mRNA can be preferentially recruited. There are 4 small upstream open reading frames (suORF) upstream of the GCN4 ORF. In order to translate the distal open reading frame of GCN4 mRNA, the 40S subunit remains bound to the mRNA after translation of the proximal suORF. With the termination of translation of the first suORF and the detachment of the 60S subunit, 50% of the 40S subunits are still bound to the mRNA and continue to scan, thereby improving translation efficiency.
After translation is terminated, the 40S subunit is still bound to the 3′-UTR of the mRNA, which is conducive to re-initiation, and the length of the 3′-UTR determines its binding time. mRNAs with low translation efficiency often use this mechanism to construct a series of mRNAs with different 3′-UTR lengths. As the 3′-UTR length increases, the translation efficiency also increases. The longer the 3′-URT, the longer ribosomes remain bound to the 3′-UTR after translation is terminated, thereby increasing their recruitment response. Moreover, during this process, the concentration of 40S subunits bound to the mRNA is higher than the concentration of the 40S subunits that have been detached from the mRNA. The PABP/polyA complex and the eIF4F/5′-end cap complex may facilitate re-recruitment [4].
2. The interaction between both ends improves mRNA stability
The interaction between PABP and CBP not only promotes efficient translation initiation, but also plays a role in maintaining the integrity of mRNA. important role [4, 9]. In yeast and mammals, polyA removal occurs before decapping when mRNA is degraded. PolyA is first degraded, causing PABP to be released from the mRNA. With the release of PABP, the 5' end cap is cleaved off by the decapping enzyme DcplP, and the entire mRNA is rapidly degraded by the 5'→3' RNA exo-ribosomal exonuclease XrnlP. The release of PABP from the mRNA leaves the 5' end cap vulnerable to attack, and PABP plays a protective role in this process. PABP can enhance the combination of plant eEF4F and the cap structure, indicating that PABP uses eIF4G as an intermediary to exert its function by stabilizing the combination of eIF4E and the cap [2]. In mammals, the removal of polyA from mRNA occurs before the degradation of the 5'-end cap, indicating that PABP may use PAIP-1 as an intermediary to promote the binding of eIF4F to the cap and exert its protective effect [4].
3. Regulation of the functional effects of both ends of mRNA
There are a variety of internal and external factors that regulate the interaction between the 5' end cap of mRNA and polyA, such as protein modifications. In mammalian cell culture, translation is inhibited during serum starvation, and conversely translation is activated. In addition, insulin can also induce the cap/polyA synergy in serum-starved cells to promote translation in a concentration-dependent manner, indicating that the regulation of the interaction between PABP and cap-related initiation factors (possibly mediated by PAIP-1) is an insulin signal transduction pathway. part of 〔11〕. The regulation of insulin may be accomplished through phosphorylation of protein factors [4]. For example, it can induce phosphorylation of eIF4E, thereby increasing its activity of binding to the cap, or inducing phosphorylation of eIF4E-binding protein, promoting the interaction between eIF4E and eIF4G, and ultimately affecting the recruitment of eIF4A, thereby affecting its interaction with PAIP-1 at both ends. The role of "bridge" between.
Gene induction is another way to regulate the functional effects of both ends. Studies have found that T cells are activated to induce the production of PAIP-1, and then PAIP-1 interacts with polyA binding protein (iPABP) [9].
Environmental stress such as heat shock can, on the one hand, rapidly disintegrate polyribosomes, and on the other hand, reduce the interaction between the cap of mRNA and polyA and inhibit translation. Heat shock directly or indirectly changes the phosphorylation status of protein factors bound to PABP, such as dephosphorylation of mammalian eIF4E and eIF4B [1] and plant eIF4B [11]. Dephosphorylation directly reduces the effects of eIF4F/eIF4B and PABP in plants; while in mammals, dephosphorylation indirectly reduces the recruitment of eIF4A and its opportunity to interact with the PAIP-1/PABP/polyA complex, thereby inhibiting translation.
4. The function of the interaction between the ends of mRNA without polyA and caps
Studies have shown that the two ends of mRNA without polyA or cap structures can also interact and play a role in translation. The mRNA of cell cycle regulatory histones in mammals does not have polyA, but has a conserved stem-loop structure at its 5' end, which is necessary for nucleocytoplasmic transport and regulation of mRNA stability during different cell cycles. It was also found that it is also required for efficient translation of mammalian mRNA terminated with a stem-loop structure. This stem-loop structure is similar to polyA, and its activity as a regulatory factor depends on the 5'-end cap, indicating that there is also an interaction between the 5'-end cap and the stem-loop structure.
Some mRNAs with polyA but no 5′-end cap have been found in viruses, such as the genomic mRNA of tomato etching virus, which uses a 5′-end leader sequence instead of the 5′-end cap to confer independence on the mRNA. Hat translation function. The 5′ leader sequence interacts with polyA like a 5′ cap to promote efficient translation. However, the protein factors that mediate this interaction and the role of both ends of cell cycle-regulated histone mRNA are still under investigation.
The two ends of some other viral RNAs lacking caps or polyA also show functional interactions, which are accomplished through RNA elements that have similar functions to caps or polyA. For example, TMV mRNA does not have polyA, but contains a 20 bp 3′-UTR, which has a similar function to polyA. This 3′-UTR is a higher-order structure containing five RNA pseudo-knots and a tRNA-like terminal region.
Research on non-conserved mRNA without polyA or cap structures suggests that sequence elements flanking the open reading frame may be the basis for efficient translation. There are still many questions about the protein translation mechanism that remain unclear. Circular mRNA translation may be one of the protein translation mechanisms, which requires further study.