AUG Selection Research Articles

Selective translational control of cellular and viral mRNAs by RPS3 mRNA binding.

RPS3, a universal core component of the 40S ribosomal subunit, interacts with mRNA at the entry channel. Whether RPS3 mRNA-binding contributes to specific mRNA translation and ribosome specialization in mammalian cells is unknown. Here we mutated RPS3 mRNA-contacting residues R116, R146 and K148 and report their impact on cellular and viral translation. R116D weakened cap-proximal initiation and promoted leaky scanning, while R146D had the opposite effect. Additionally, R146D and K148D displayed contrasting effects on start-codon fidelity. Translatome analysis uncovered common differentially translated genes of which the downregulated set bears long 5'UTR and weak AUG context, suggesting a stabilizing role during scanning and AUG selection. We identified an RPS3-dependent regulatory sequence (RPS3RS) in the sub-genomic 5'UTR of SARS-CoV-2 consisting of a CUG initiation codon and a downstream element that is also the viral transcription regulatory sequence (TRS). Furthermore, RPS3 mRNA-binding residues are essential for SARS-CoV-2 NSP1-mediated inhibition of host translation and for its ribosomal binding. Intriguingly, NSP1-induced mRNA degradation was also reduced in R116D cells, indicating that mRNA decay occurs in the ribosome context. Thus, RPS3 mRNA-binding residues have multiple translation regulatory functions and are exploited by SARS-CoV-2 in various ways to influence host and viral mRNA translation and stability.

Identification of Selected Kinetoplastids 18S rRNA Residues required for Efficient Recruitment of Initiator tRNA Met and AUG Selection in silico

High Resolution 18S rRNA structures of kinetoplastids ribosomes from theoretical methods have provided atomic level details about the process of translation. This process entails detailed information on the mRNA and tRNA binding and decoding centers within the 18S rRNA that was previously not very well understood. We identified residues in selected kinetoplastids 18S rRNA critical in recruiting the first methionyl tRNA to the small ribosome subunit during initiation and comparing them to see the differences. The Kozak sequence presence on eukaryotic mRNAs tethers it to the AUG start codon. Kinetoplastids are a closely related group, and the three chosen exhibited differences in the A-site in terms of position and nucleotides found there. Interactions are found at the A-site (543-UUU-546 for T. cruzi, 560-CCUA-563 for T. brucei, and 540-UUUG-543 for Leishmania major), where the different mRNA get complementary sequences at the 16th helix. The current findings show that each messenger RNA has a sequence that is complementary to the appropriate 18S rRNA sequence, tethering the mRNA to the small ribosomal subunit, which then recruits the bigger subunit. When compared to the Kozak region that flanks the AUG start codon, this method effectively promotes start codon placement.

Altered proteome in translation initiation fidelity defective eIF5G31R mutant causes oxidative stress and DNA damage

The recognition of the AUG start codon and selection of an open reading frame (ORF) is fundamental to protein biosynthesis. Defect in the fidelity of start codon selection adversely affect proteome and have a pleiotropic effect on cellular function. Using proteomic techniques, we identified differential protein abundance in the translation initiation fidelity defective eIF5G31R mutant that initiates translation using UUG codon in addition to the AUG start codon. Consistently, the eIF5G31R mutant altered proteome involved in protein catabolism, nucleotide biosynthesis, lipid biosynthesis, carbohydrate metabolism, oxidation–reduction pathway, autophagy and re-programs the cellular pathways. The utilization of the upstream UUG codons by the eIF5G31R mutation caused downregulation of uridylate kinase expression, sensitivity to hydroxyurea, and DNA damage. The eIF5G31R mutant cells showed lower glutathione levels, high ROS activity, and sensitivity to H2O2.

Cancer-Associated Eukaryotic Translation Initiation Factor 1A Mutants Impair Rps3 and Rps10 Binding and Enhance Scanning of Cell Cycle Genes.

Protein synthesis is linked to cell proliferation, and its deregulation contributes to cancer. Eukaryotic translation initiation factor 1A (eIF1A) plays a key role in scanning and AUG selection and differentially affects the translation of distinct mRNAs. Its unstructured N-terminal tail (NTT) is frequently mutated in several malignancies. Here we report that eIF1A is essential for cell proliferation and cell cycle progression. Ribosome profiling of eIF1A knockdown cells revealed a substantial enrichment of cell cycle mRNAs among the downregulated genes, which are predominantly characterized by a lengthy 5' untranslated region (UTR). Conversely, eIF1A depletion caused a broad stimulation of 5' UTR initiation at a near cognate AUG, unveiling a prominent role of eIF1A in suppressing 5' UTR translation. In addition, the AUG context-dependent autoregulation of eIF1 was disrupted by eIF1A depletion, suggesting their cooperation in AUG context discrimination and scanning. Importantly, cancer-associated eIF1A NTT mutants augmented the eIF1A positive effect on a long 5' UTR, while they hardly affected AUG selection. Mechanistically, these mutations diminished the eIF1A interaction with Rps3 and Rps10 implicated in scanning arrest. Our findings suggest that the reduced binding of eIF1A NTT mutants to the ribosome retains its open state and facilitates scanning of long 5' UTR-containing cell cycle genes.

Translational initiation factor eIF5 replaces eIF1 on the 40S ribosomal subunit to promote start-codon recognition.

In eukaryotic translation initiation, AUG recognition of the mRNA requires accommodation of Met-tRNAi in a 'PIN' state, which is antagonized by the factor eIF1. eIF5 is a GTPase activating protein (GAP) of eIF2 that additionally promotes stringent AUG selection, but the molecular basis of its dual function was unknown. We present a cryo-electron microscopy (cryo-EM) reconstruction of a yeast 48S pre-initiation complex (PIC), at an overall resolution of 3.0 Å, featuring the N-terminal domain (NTD) of eIF5 bound to the 40S subunit at the location vacated by eIF1. eIF5 interacts with and allows a more accommodated orientation of Met-tRNAi. Substitutions of eIF5 residues involved in the eIF5-NTD/tRNAi interaction influenced initiation at near-cognate UUG codonsin vivo, and the closed/open PIC conformation in vitro, consistent with direct stabilization of the codon:anticodon duplex by the wild-type eIF5-NTD. The present structure reveals the basis for a key role of eIF5 in start-codon selection.

Dynamic Interaction of Eukaryotic Initiation Factor 4G1 (eIF4G1) with eIF4E and eIF1 Underlies Scanning-Dependent and -Independent Translation.

Translation initiation of most mRNAs involves m7G-cap binding, ribosomal scanning, and AUG selection. Initiation from an m7G-cap-proximal AUG can be bypassed resulting in leaky scanning, except for mRNAs bearing the translation initiator of short 5' untranslated region (TISU) element. m7G-cap binding is mediated by the eukaryotic initiation factor 4E (eIF4E)-eIF4G1 complex. eIF4G1 also associates with eIF1, and both promote scanning and AUG selection. Understanding of the dynamics and significance of these interactions is lacking. We report that eIF4G1 exists in two complexes, either with eIF4E or with eIF1. Using an eIF1 mutant impaired in eIF4G1 binding, we demonstrate that eIF1-eIF4G1 interaction is important for leaky scanning and for avoiding m7G-cap-proximal initiation. Intriguingly, eIF4E-eIF4G1 antagonizes the scanning promoted by eIF1-eIF4G1 and is required for TISU. In mapping the eIF1-binding site on eIF4G1, we unexpectedly found that eIF4E also binds it indirectly. These findings uncover the RNA features underlying regulation by eIF4E-eIF4G1 and eIF1-eIF4G1 and suggest that 43S ribosome transition from the m7G-cap to scanning involves relocation of eIF4G1 from eIF4E to eIF1.

Crystal Structure of ATP-Bound Human ABCF1 Demonstrates a Unique Conformation of ABC Proteins

Gene translation requires the correct selection of start codon AUG in mRNA. ATP-binding cassette subfamily F member 1 (ABCF1) plays a key role in the accuracy of start codon selection. However, the function of human ABCF1 is not clearly understood. Here, we solve the crystal structure of an ATP-bound wild-type human ABCF1 at 2.3-Å resolution. The comparative studies indicate that the structure is in a pre-activation intermediate conformation. This conformation is stabilized by the interaction between ATP and protein. Thus, we propose that this conformation is an important step in the activation of ABCF1. This study extends our understanding of ABC (ATP-binding cassette) protein activation at the molecular level.

Interface between 40S exit channel protein uS7/Rps5 and eIF2α modulates start codon recognition in vivo.

The eukaryotic pre-initiation complex (PIC) bearing the eIF2·GTP·Met-tRNAiMet ternary complex (TC) scans the mRNA for an AUG codon in favorable context. AUG recognition evokes rearrangement of the PIC from an open, scanning to a closed, arrested conformation. Cryo-EM reconstructions of yeast PICs suggest remodeling of the interface between 40S protein Rps5/uS7 and eIF2α between open and closed states; however, its importance was unknown. uS7 substitutions disrupting eIF2α contacts favored in the open complex increase initiation at suboptimal sites, and uS7-S223D stabilizes TC binding to PICs reconstituted with a UUG start codon, indicating inappropriate rearrangement to the closed state. Conversely, uS7-D215 substitutions, perturbing uS7-eIF2α interaction in the closed state, confer the opposite phenotypes of hyperaccuracy and (for D215L) accelerated TC dissociation from reconstituted PICs. Thus, remodeling of the uS7/eIF2α interface appears to stabilize first the open, and then the closed state of the PIC to promote accurate AUG selection in vivo.

The β-hairpin of 40S exit channel protein Rps5/uS7 promotes efficient and accurate translation initiation in vivo.

The eukaryotic 43S pre-initiation complex bearing tRNAi(Met) scans the mRNA leader for an AUG start codon in favorable context. Structural analyses revealed that the β-hairpin of 40S protein Rps5/uS7 protrudes into the 40S mRNA exit-channel, contacting the eIF2∙GTP∙Met-tRNAi ternary complex (TC) and mRNA context nucleotides; but its importance in AUG selection was unknown. We identified substitutions in β-strand-1 and C-terminal residues of yeast Rps5 that reduced bulk initiation, conferred 'leaky-scanning' of AUGs; and lowered initiation fidelity by exacerbating the effect of poor context of the eIF1 AUG codon to reduce eIF1 abundance. Consistently, the β-strand-1 substitution greatly destabilized the 'PIN' conformation of TC binding to reconstituted 43S·mRNA complexes in vitro. Other substitutions in β-hairpin loop residues increased initiation fidelity and destabilized PIN at UUG, but not AUG start codons. We conclude that the Rps5 β-hairpin is as crucial as soluble initiation factors for efficient and accurate start codon recognition.

C/EBPα in normal and malignant myelopoiesis.

CCAAT/enhancer binding protein α (C/EBPα) dimerizes via its leucine zipper (LZ) domain to bind DNA via its basic region and activate transcription via N-terminal trans-activation domains. The activity of C/EBPα is modulated by several serine/threonine kinases and via sumoylation, its gene is activated by RUNX1 and additional transcription factors, its mRNA stability is modified by miRNAs, and its mRNA is subject to translation control that affects AUG selection. In addition to inducing differentiation, C/EBPα inhibits cell cycle progression and apoptosis. Within hematopoiesis, C/EBPα levels increase as long-term stem cells progress to granulocyte-monocyte progenitors (GMP). Absence of C/EBPα prevents GMP formation, and higher levels are required for granulopoiesis compared to monopoiesis. C/EBPα interacts with AP-1 proteins to bind hybrid DNA elements during monopoiesis, and induction of Gfi-1, C/EBPε, KLF5, and miR-223 by C/EBPα enables granulopoiesis. The CEBPA ORF is mutated in approximately 10% of acute myeloid leukemias (AML), leading to expression of N-terminally truncated C/EBPαp30 and C-terminal, in-frame C/EBPαLZ variants, which inhibit C/EBPα activities but also play additional roles during myeloid transformation. RUNX1 mutation, CEBPA promoter methylation, Trib1 or Trib2-mediated C/EBPαp42 degradation, and signaling pathways leading to C/EBPα serine 21 phosphorylation reduce C/EBPα expression or activity in additional AML cases.

Translation of foot-and-mouth disease virus RNA: factors influencing alternative AUG selection.

The mechanism of alternative AUG usage in foot-and-mouth disease virus is not completely understood. Using simple computational approaches, we evaluated the contributions of overall codon bias, quantitative codon bias, and %GC of the region between the two alternative AUGs, Region-La, as well as the nucleotide bias of the sequence context flanking each AUG with respect to translation initiation efficiency. For all serotypes of this virus, we found that only a small component of the effect of RNA secondary structure on ribosome scanning was due to the low %GC of Region-La. In addition, we found that the nucleotide bias of the context from position -4 to +6 flanking the AUG(2nd) had a negative correlation with the overall codon bias, and that a strong purine bias existed in this AUG(2nd)context. However, the quantitative codon bias of Region-La was seen to be significantly lower than that of Region-Lb (the sequence following AUG(2nd)) in all serotypes except SAT 1-3. Taken together, our results suggest that the low codon bias of Region-La might impair the translation initiation efficiency at the AUG(1st) in all serotypes except SAT 1-3, and the specific AUG(2nd) context might be used as a strong signal to initiate translation from the AUG(2nd) in all serotypes.

Initiation codon selection is accomplished by a scanning mechanism without crucial initiation factors in Sindbis virus subgenomic mRNA.

Translation initiation of alphavirus subgenomic mRNA (sgmRNA) can occur in the absence of several initiation factors (eIFs) in infected cells; however, the precise translation mechanism is still poorly understood. In this study, we have examined the mechanism of initiation and AUG selection in Sindbis virus (SINV) sgmRNA. Our present findings suggest that sgmRNA is translated via a scanning mechanism, since the presence of a hairpin structure before the initiation codon hampers protein synthesis directed by this mRNA. In addition, translation is partially recovered when an in-frame AUG codon is placed upstream of this hairpin. This scanning process takes place without the participation of eIF4A and active eIF2. These results, combined with our findings through modifying the SINV sgmRNA leader sequence, do not support the possibility of a direct initiation from the start codon without previous scanning, or a shunting mechanism. Moreover, studies carried out with sgmRNAs containing two alternative AUG codons within a good context for translation reveal differences in AUG selection which are dependent on the cellular context and the phosphorylation state of eIF2α. Thus, initiation at the additional AUG is strictly dependent on active eIF2, whereas the genuine AUG codon can start translation following eIF2α inactivation. Collectively, our results suggest that SINV sgmRNA is translated by a scanning mechanism without the potential participation of crucial eIFs. A model is presented that explains the mechanism of initiation of mRNAs bearing two alternative initiation codons.

Eukaryotic translation initiation factor eIF5 promotes the accuracy of start codon recognition by regulating Pi release and conformational transitions of the preinitiation complex.

eIF5 is the GTPase activating protein (GAP) for the eIF2·GTP·Met-tRNAiMet ternary complex with a critical role in initiation codon selection. Previous work suggested that the eIF5 mutation G31R/SUI5 elevates initiation at UUG codons by increasing GAP function. Subsequent work implicated eIF5 in rearrangement of the preinitiation complex (PIC) from an open, scanning conformation to a closed state at AUG codons, from which Pi is released from eIF2·GDP·Pi. To identify eIF5 functions crucial for accurate initiation, we investigated the consequences of G31R on GTP hydrolysis and Pi release, and the effects of intragenic G31R suppressors on these reactions, and on the partitioning of PICs between open and closed states. eIF5-G31R altered regulation of Pi release, accelerating it at UUG while decreasing it at AUG codons, consistent with its ability to stabilize the closed complex at UUG. Suppressor G62S mitigates both defects of G31R, accounting for its efficient suppression of UUG initiation in G31R,G62S cells; however suppressor M18V impairs GTP hydrolysis with little effect on PIC conformation. The strong defect in GTP hydrolysis conferred by M18V likely explains its broad suppression of Sui− mutations in numerous factors. We conclude that both of eIF5's functions, regulating Pi release and stabilizing the closed PIC conformation, contribute to stringent AUG selection in vivo.

The Interaction between Eukaryotic Initiation Factor 1A and eIF5 Retains eIF1 within Scanning Preinitiation Complexes

Scanning of the mRNA transcript by the preinitiation complex (PIC) requires a panel of eukaryotic initiation factors, which includes eIF1 and eIF1A, the main transducers of stringent AUG selection. eIF1A plays an important role in start codon recognition; however, its molecular contacts with eIF5 are unknown. Using nuclear magnetic resonance, we unveil eIF1A's binding surface on the carboxyl-terminal domain of eIF5 (eIF5-CTD). We validated this interaction by observing that eIF1A does not bind to an eIF5-CTD mutant, altering the revealed eIF1A interaction site. We also found that the interaction between eIF1A and eIF5-CTD is conserved between humans and yeast. Using glutathione S-transferase pull-down assays of purified proteins, we showed that the N-terminal tail (NTT) of eIF1A mediates the interaction with eIF5-CTD and eIF1. Genetic evidence indicates that overexpressing eIF1 or eIF5 suppresses the slow growth phenotype of eIF1A-NTT mutants. These results suggest that the eIF1A-eIF5-CTD interaction during scanning PICs contributes to the maintenance of eIF1 within the open PIC.

Interaction between 25S rRNA A Loop and Eukaryotic Translation Initiation Factor 5B Promotes Subunit Joining and Ensures Stringent AUG Selection

In yeast, 25S rRNA makes up the major mass and shape of the 60S ribosomal subunit. During the last step of translation initiation, eukaryotic initiation factor 5B (eIF5B) promotes the 60S subunit joining with the 40S initiation complex (IC). Malfunctional 60S subunits produced by misfolding or mutation may disrupt the 40S IC stalling on the start codon, thereby altering the stringency of initiation. Using several point mutations isolated by random mutagenesis, here we studied the role of 25S rRNA in start codon selection. Three mutations changing bases near the ribosome surface had strong effects, allowing the initiating ribosomes to skip both AUG and non-AUG codons: C2879U and U2408C, altering the A loop and P loop, respectively, of the peptidyl transferase center, and G1735A, mapping near a Eukarya-specific bridge to the 40S subunit. Overexpression of eIF5B specifically suppressed the phenotype caused by C2879U, suggesting functional interaction between eIF5B and the A loop. In vitro reconstitution assays showed that C2879U decreased eIF5B-catalyzed 60S subunit joining with a 40S IC. Thus, eIF5B interaction with the peptidyl transferase center A loop increases the accuracy of initiation by stabilizing the overall conformation of the 80S initiation complex. This study provides an insight into the effect of ribosomal mutations on translation profiles in eukaryotes.

β-Hairpin Loop of Eukaryotic Initiation Factor 1 (eIF1) Mediates 40 S Ribosome Binding to Regulate Initiator tRNAMet Recruitment and Accuracy of AUG Selection in Vivo

Recognition of the translation initiation codon is thought to require dissociation of eIF1 from the 40 S ribosomal subunit, enabling irreversible GTP hydrolysis (Pi release) by the eIF2·GTP·Met-tRNAi ternary complex (TC), rearrangement of the 40 S subunit to a closed conformation incompatible with scanning, and stable binding of Met-tRNAi to the P site. The crystal structure of a Tetrahymena 40 S·eIF1 complex revealed several basic amino acids in eIF1 contacting 18 S rRNA, and we tested the prediction that their counterparts in yeast eIF1 are required to prevent premature eIF1 dissociation from scanning ribosomes at non-AUG triplets. Supporting this idea, substituting Lys-60 in helix α1, or either Lys-37 or Arg-33 in β-hairpin loop-1, impairs binding of yeast eIF1 to 40 S·eIF1A complexes in vitro, and it confers increased initiation at UUG codons (Sui(-) phenotype) or lethality, in a manner suppressed by overexpressing the mutant proteins or by an eIF1A mutation (17-21) known to impede eIF1 dissociation in vitro. The eIF1 Sui(-) mutations also derepress translation of GCN4 mRNA, indicating impaired ternary complex loading, and this Gcd(-) phenotype is likewise suppressed by eIF1 overexpression or the 17-21 mutation. These findings indicate that direct contacts of eIF1 with 18 S rRNA seen in the Tetrahymena 40 S·eIF1 complex are crucial in yeast to stabilize the open conformation of the 40 S subunit and are required for rapid TC loading and ribosomal scanning and to impede rearrangement to the closed complex at non-AUG codons. Finally, we implicate the unstructured N-terminal tail of eIF1 in blocking rearrangement to the closed conformation in the scanning preinitiation complex.

Unique translation initiation of mRNAs-containing TISU element

Translation Initiator of Short 5′ UTR (TISU) is a unique regulatory element of both transcription and translation initiation. It is present in a sizable number of genes with basic cellular functions and a very short untranslated region (5′ UTR). Here, we investigated translation initiation from short 5′ UTR mRNAs with AUG in various contexts. Reducing 5′ UTR length to the minimal functional size increases leaky scanning from weak and strong initiators but hardly affects translation initiation and ribosomal binding directed by TISU. Ribosome interaction with TISU mRNA is cap dependent and involves AUG downstream nucleotides that compensate for the absent 5′ UTR contacts. Interestingly, eIF1 inhibits cap-proximal AUG selection within weak or strong contexts but not within TISU. Furthermore, TISU-directed translation is unaffected by inhibition of the RNA helicase eIF4A. Thus, TISU directs efficient cap-dependent translation initiation without scanning, a mechanism that would be advantageous when intracellular levels of eIF1 and eIF4A fluctuate.

Yeast 18 S rRNA Is Directly Involved in the Ribosomal Response to Stringent AUG Selection during Translation Initiation

In eukaryotes, the 40 S ribosomal subunit serves as the platform of initiation factor assembly, to place itself precisely on the AUG start codon. Structural arrangement of the 18 S rRNA determines the overall shape of the 40 S subunit. Here, we present genetic evaluation of yeast 18 S rRNA function using 10 point mutations altering the polysome profile. All the mutants reduce the abundance of the mutant 40 S, making it limiting for translation initiation. Two of the isolated mutations, G875A, altering the core of the platform domain that binds eIF1 and eIF2, and A1193U, changing the h31 loop located below the P-site tRNAiMet, show phenotypes indicating defective regulation of AUG selection. Evidence is provided that these mutations reduce the interaction with the components of the preinitiation complex, thereby inhibiting its function at different steps. These results indicate that the 18 S rRNA mutations impair the integrity of scanning-competent preinitiation complex, thereby altering the 40 S subunit response to stringent AUG selection. Interestingly, nine of the mutations alter the body/platform domains of 18 S rRNA, potentially affecting the bridges to the 60 S subunit, but they do not change the level of 18 S rRNA intermediates. Based on these results, we also discuss the mechanism of the selective degradation of the mutant 40 S subunits.

The Eukaryotic Initiation Factor (eIF) 4G HEAT Domain Promotes Translation Re-initiation in Yeast Both Dependent on and Independent of eIF4A mRNA Helicase

Translation re-initiation provides the molecular basis for translational control of mammalian ATF4 and yeast GCN4 mediated by short upstream open reading (uORFs) in response to eIF2 phosphorylation. eIF4G is the major adaptor subunit of eIF4F that binds the cap-binding subunit eIF4E and the mRNA helicase eIF4A and is also required for re-initiation in mammals. Here we show that the yeast eIF4G2 mutations altering eIF4E- and eIF4A-binding sites increase re-initiation at GCN4 and impair recognition of the start codons of uORF1 or uORF4 located after uORF1. The increase in re-initiation at GCN4 was partially suppressed by increasing the distance between uORF1 and GCN4, suggesting that the mutations decrease the migration rate of the scanning ribosome in the GCN4 leader. Interestingly, eIF4E overexpression suppressed both the phenotypes caused by the mutation altering eIF4E-binding site. Thus, eIF4F is required for accurate AUG selection and re-initiation also in yeast, and the eIF4G interaction with the mRNA-cap appears to promote eIF4F re-acquisition by the re-initiating 40 S subunit. However, eIF4A overexpression suppressed the impaired AUG recognition but not the increase in re-initiation caused by the mutations altering eIF4A-binding site. These results not only provide evidence that mRNA unwinding by eIF4A stimulates start codon recognition, but also suggest that the eIF4A-binding site on eIF4G made of the HEAT domain stimulates the ribosomal scanning independent of eIF4A. Based on the RNA-binding activities identified within the unstructured segments flanking the eIF4G2 HEAT domain, we discuss the role of the HEAT domain in scanning beyond loading eIF4A onto the pre-initiation complex.

Characteristics of codon usage bias in two regions downstream of the initiation codons of foot-and-mouth disease virus

The mechanism of utilization of alternative two AUGs in foot-and-mouth disease virus (FMDV) is still unknown to date. In this study, the characteristics of codon usage bias (CUB) of the region between the two AUGs (the region-La) and of the same-sized region behind the second AUG (the region-Lb) in 94 different FMDV RNA sequences were analyzed using relative synonymous codon usage (RSCU) values. The results indicated that many codons with negative CUB were preferentially used in the region-La. There were two conserved residues (Thr and Cys) on the 4th and 6th residue positions of the region-La. The conserved residues had a general tendency to choose synonymous codons with negative CUB. Although most positions in the region-La did not contain conserved residues, many positions tended to use codons with negative CUB in this region. Among these codons, the majority belonged to the amino acids containing synonymous codons with clearly positive and negative CUB, including Asp, Val, Ile, Leu, Thr, Ala, Ser, Asn and Arg. The presence of many codons with negative CUB in the region-La might impair the efficiency of the first AUG selection. The phylogenetic incongruence of the region-La and the region-Lb implied that intertypic recombination played an important role in the evolution of FMDV. Furthermore, due to the existence of more positions with positive CUB and more widespread phylogenetic incongruence in the region-Lb than the region-La, a probable relationship between the degree of CUB and the evolution of the two target regions was revealed.