DeepCIP: A multimodal deep learning method for the prediction of internal ribosome entry sites of circRNAs.

Circular RNAs (circRNAs) have been found to have the ability to encode proteins through internal ribosome entry sites (IRESs), which are essential RNA regulatory elements for cap-independent translation. Identification of IRES elements in circRNA is crucial for understanding its function. Previous studies have presented IRES predictors based on machine learning techniques, but they were mainly designed for linear RNA IRES. In this study, we proposed DeepCIP (Deep learning method for CircRNA IRES Prediction), a multimodal deep learning approach that employs both sequence and structural information for circRNA IRES prediction. Our results demonstrate the effectiveness of the sequence and structure models used by DeepCIP in feature extraction and suggest that integrating sequence and structural information efficiently improves the accuracy of prediction. The comparison studies indicate that DeepCIP outperforms other comparative methods on the test set and real circRNA IRES dataset. Furthermore, through the integration of an interpretable analysis mechanism, we elucidate the sequence patterns learned by our model, which align with the previous discovery of motifs that facilitate circRNA translation. Thus, DeepCIP has the potential to enhance the study of the coding potential of circRNAs and contribute to the design of circRNA-based drugs. DeepCIP as a standalone program is freely available at https://github.org/zjupgx/DeepCIP.

Function-Related Asymmetry of the Interactions between Matrix Loops and Conserved Sequence Motifs in the Mitochondrial ADP/ATP Carrier.

The ADP/ATP carrier (AAC) plays a central role in oxidative metabolism by exchanging ATP and ADP across the inner mitochondrial membrane. Previous experiments have shown the involvement of the matrix loops of AAC in its function, yet potential mechanisms remain largely elusive. One obstacle is the limited information on the structural dynamics of the matrix loops. In the current work, unbiased all-atom molecular dynamics (MD) simulations were carried out on c-state wild-type AAC and mutants. Our results reveal that: (1) two ends of a matrix loop are tethered through interactions between the residue of triplet 38 (Q38, D143 and Q240) located at the C-end of the odd-numbered helix and residues of the [YF]xG motif located before the N-end of the short matrix helix in the same domain; (2) the initial progression direction of a matrix loop is determined by interactions between the negatively charged residue of the [DE]G motif located at the C-end of the short matrix helix and the capping arginine (R30, R139 and R236) in the previous domain; (3) the two chemically similar residues D and E in the highly conserved [DE]G motif are actually quite different; (4) the N-end of the M3 loop is clamped by the [DE]G motif and the capping arginine of domain 2 from the two sides, which strengthens interactions between domain 2 and domain 3; and (5) a highly asymmetric stable core exists within domains 2 and 3 at the m-gate level. Moreover, our results help explain almost all extremely conserved residues within the matrix loops of the ADP/ATP carriers from a structural point of view. Taken together, the current work highlights asymmetry in the three matrix loops and implies a close relationship between asymmetry and ADP/ATP transport.

The effects of cardiolipin on the structural dynamics of the mitochondrial ADP/ATP carrier in its cytosol-open state.

Cardiolipin (CL) has been shown to play a crucial role in regulating the function of proteins in the inner mitochondrial membrane. As the most abundant protein of the inner mitochondrial membrane, the ADP/ATP carrier (AAC) has long been the model of choice to study CL-protein interactions, and specifically bound CLs have been identified in a variety of crystal structures of AAC. However, how CL binding affects the structural dynamics of AAC in atomic detail remains largely elusive. Here we compared all-atom molecular dynamics simulations on bovine AAC1 in lipid bilayers with and without CLs. Our results show that on the current microsecond simulation time scale: 1) CL binding does not significantly affect overall stability of the carrier or structural symmetry at the matrix-gate level; 2) pocket volumes of the carrier and interactions involved in the matrix-gate network become more heterogeneous in parallel simulations with membranes containing CLs; 3) CL binding consistently strengthens backbone hydrogen bonds within helix H2 near the matrix side; and 4) CLs play a consistent stabilizing role on the domain 1-2 interface through binding with the R30:R71:R151 stacking structure and fixing the M2 loop in a defined conformation. CL is necessary for the formation of this stacking structure, and this structure in turn forms a very stable CL binding site. Such a delicate equilibrium suggests the strictly conserved R30:R71:R151stacking structure of AACs could function as a switch under regulation of CLs. Taken together, these results shed new light on the CL-mediated modulation of AAC function.

Mechanistic insights into multiple-step transport of mitochondrial ADP/ATP carrier.

The ADP/ATP carrier (AAC) is crucial for mitochondrial functions by importing ADP and exporting ATP across the inner mitochondrial membrane. However, the mechanism of highly specific ADP recognition and transport by AAC remains largely elusive. In this work, spontaneous ADP binding process to the ground c-state AAC was investigated through rigorous molecular dynamics simulations of over 31 microseconds in total. With improved simulation strategy, we have successfully identified a highly specific ADP binding site in the upper region of the cavity, and this site exhibits selectivity for ADP over ATP based on free-energy calculations. Sequence analyses on adenine nucleotide transporters also suggest that this subgroup uses the upper region of the cavity, rather than the previously proposed central binding site located at the bottom of the cavity to discriminate their substrates. Identification of the new site unveils the unusually high substrate specificity of AAC and explains the dependence of transport on the flexibility between anti and syn glycosidic conformers of ADP. Moreover, this new site together with the central site supports early biochemical findings. In light of these early findings, our simulations described a multi-step model in which the carrier uses different sites for substrate attraction, recognition and conformational transition. These results provide new insights into the transport mechanism of AAC and other adenine nucleotide transporters.

Investigating the Broad Matrix-Gate Network in the Mitochondrial ADP/ATP Carrier through Molecular Dynamics Simulations.

The mitochondrial ADP/ATP carrier (AAC) exports ATP and imports ADP through alternating between cytosol-open (c-) and matrix-open (m-) states. The salt bridge networks near the matrix side (m-gate) and cytosol side (c-gate) are thought to be crucial for state transitions, yet our knowledge on these networks is still limited. In the current work, we focus on more conserved m-gate network in the c-state AAC. All-atom molecular dynamics (MD) simulations on a variety of mutants and the CATR-AAC complex have revealed that: (1) without involvement of other positive residues, the charged residues from the three Px[DE]xx[KR] motifs only are prone to form symmetrical inter-helical network; (2) R235 plays a determinant role for the asymmetry in m-gate network of AAC; (3) R235 significantly strengthens the interactions between H3 and H5; (4) R79 exhibits more significant impact on m-gate than R279; (5) CATR promotes symmetry in m-gate mainly through separating R234 from D231 and fixing R79; (6) vulnerability of the H2-H3 interface near matrix side could be functionally important. Our results provide new insights into the highly conserved yet variable m-gate network in the big mitochondrial carrier family.

Ablation of Mto1 in zebrafish exhibited hypertrophic cardiomyopathy manifested by mitochondrion RNA maturation deficiency.

Deficient maturations of mitochondrial transcripts are linked to clinical abnormalities but their pathophysiology remains elusive. Previous investigations showed that pathogenic variants in MTO1 for the biosynthesis of τm5U of tRNAGlu, tRNAGln, tRNALys, tRNATrp and tRNALeu(UUR) were associated with hypertrophic cardiomyopathy (HCM). Using mto1 knock-out(KO) zebrafish generated by CRISPR/Cas9 system, we demonstrated the pleiotropic effects of Mto1 deficiency on mitochondrial RNA maturations. The perturbed structure and stability of tRNAs caused by mto1 deletion were evidenced by conformation changes and sensitivity to S1-mediated digestion of tRNAGln, tRNALys, tRNATrp and tRNALeu(UUR). Notably, mto1KO zebrafish exhibited the global decreases in the aminoacylation of mitochondrial tRNAs with the taurine modification. Strikingly, ablated mto1 mediated the expression of MTPAP and caused the altered polyadenylation of cox1, cox3, and nd1 mRNAs. Immunoprecipitation assay indicated the interaction of MTO1 with MTPAP related to mRNA polyadenylation. These alterations impaired mitochondrial translation and reduced activities of oxidative phosphorylation complexes. These mitochondria dysfunctions caused heart development defects and hypertrophy of cardiomyocytes and myocardial fiber disarray in ventricles. These cardiac defects in the mto1KO zebrafish recapitulated the clinical phenotypes in HCM patients carrying the MTO1 mutation(s). Our findings highlighted the critical role of MTO1 in mitochondrial transcript maturation and their pathological consequences in hypertrophic cardiomyopathy.

Function-related asymmetry of the specific cardiolipin binding sites on the mitochondrial ADP/ATP carrier.

The ADP/ATP carrier (AAC) transports matrix ATP and cytosolic ADP across the inner mitochondrial membrane (IMM). It is well known that cardiolipin (CL) plays an important role in regulating the function of AAC, yet the underlying mechanism still remains elusive. AAC is composed of three homologous domains, and three specific CL binding sites are located at the domain-domain interfaces near the matrix side. Here we report an in-depth investigation on the dynamic properties of the bound CL within the three specific sites through all-atom molecular dynamics simulations of up to 13 µs in total. Our results highlight the importance of the basic and polar residues in CL binding. The basic residues from the linker helix and/or the [Y/W/F][K/R]G motif enable the bound CL to form an intra-domain binding mode, and the canonical inter-domain binding mode only forms when these basic residues are occupied by an additional phospholipid. Of special significance, differences in the basic and polar residues lead to remarkable asymmetry among the three specific CL binding sites. We found that the bound CL at the interface of domains 2 and 3 predominantly adopts inter-domain binding mode, while CLs at the other two sites have much more intra-domain populations. This is consistent with the asymmetric crystal structure of the matrix state (m-state) AAC which implies an asymmetric transport mechanism. The dynamic equilibrium between the inter-domain and intra-domain binding modes observed in our simulations could be highly important for the bound CLs to adapt to the movements during state transitions.

Molecular dynamics simulations on apo ADP/ATP carrier shed new lights on the featured motif of the mitochondrial carriers.

The ADP/ATP carrier (AAC) is a transporter responsible for the equal molar exchange of cytosolic ADP and ATP synthesized within mitochondrial matrix across the mitochondrial membrane. Its primary structure consists of three homologous repeats, and each repeat contains a conserved motif that is shared by all members of the mitochondrial carrier family (MCF). Although these MCF motif residues cluster together in the crystal structure of AAC, detailed analyses on the interactions among the motif residues are still limited. In the present study, all-atom molecular dynamics (MD) simulations of up to 10 µs have been carried out on AAC, and interactions and structural dynamics of the MCF motif residues have been specifically investigated. Our simulations have revealed: i) a very asymmetrical electrostatic network at the bottom of the pocket of apo AAC, ii) the asymmetrical interactions between the Pro kink region and the [YWF][KR] G motif in three repeats, iii) the role of the conserved Arg residues in stabilizing the C-ends of the odd-numbered helices, iv) the structural change of the [YWF][KR] G motif and its potential involvement in substrate translocation process. Our results highlight the asymmetry of the MCF residues in the three repeats, which might contribute to the ability of the carriers to transport the asymmetrical substrates. Our observations provide microscopic basis for further research on the translocation mechanism of mitochondrial carriers.

Deletion of Gtpbp3 in zebrafish revealed the hypertrophic cardiomyopathy manifested by aberrant mitochondrial tRNA metabolism.

GTPBP3 is a highly conserved tRNA modifying enzyme for the biosynthesis of τm5U at the wobble position of mitochondrial tRNAGlu, tRNAGln, tRNALys, tRNATrp and tRNALeu(UUR). The previous investigations showed that GTPBP3 mutations were associated with hypertrophic cardiomyopathy (HCM). However, the pathophysiology of GTPBP3 deficiency remains elusively. Using the gtpbp3 knockout zebrafish generated by CRISPR/Cas9 system, we demonstrated the aberrant mitochondrial tRNA metabolism in gtpbp3 knock-out zebrafish. The deletion of gtpbp3 may alter functional folding of tRNA, indicated by conformation changes and sensitivity to S1-mediated digestion of tRNAGlu, tRNALys, tRNATrp and tRNALeu(UUR). Strikingly, gtpbp3 knock-out zebrafish displayed the global increases in the aminoacylated efficiencies of mitochondrial tRNAs. The aberrant mitochondrial tRNA metabolisms impaired mitochondrial translation, produced proteostasis stress and altered activities of respiratory chain complexes. These mitochondria dysfunctions caused the alterations in the embryonic heart development and reduced fractional shortening of ventricles in mutant zebrafish. Notably, the gtpbp3 knock-out zebrafish exhibited hypertrophy of cardiomyocytes and myocardial fiber disarray in ventricles. These cardiac defects in the gtpbp3 knock-out zebrafish recapitulated the clinical phenotypes in HCM patients carrying the GTPBP3 mutation(s). Our findings highlight the fundamental role of defective nucleotide modifications of tRNAs in mitochondrial biogenesis and their pathological consequences in hypertrophic cardiomyopathy.

Deletion of Mtu1 (Trmu) in zebrafish revealed the essential role of tRNA modification in mitochondrial biogenesis and hearing function.

Mtu1(Trmu) is a highly conserved tRNA modifying enzyme responsible for the biosynthesis of τm5s2U at the wobble position of tRNAGln, tRNAGlu and tRNALys. Our previous investigations showed that MTU1 mutation modulated the phenotypic manifestation of deafness-associated mitochondrial 12S rRNA mutation. However, the pathophysiology of MTU1 deficiency remains poorly understood. Using the mtu1 knock-out zebrafish generated by CRISPR/Cas9 system, we demonstrated the abolished 2-thiouridine modification of U34 of mitochondrial tRNALys, tRNAGlu and tRNAGln in the mtu1 knock-out zebrafish. The elimination of this post-transcriptional modification mediated mitochondrial tRNA metabolisms, causing the global decreases in the levels of mitochondrial tRNAs. The aberrant mitochondrial tRNA metabolisms led to the impairment of mitochondrial translation, respiratory deficiencies and reductions of mitochondrial ATP production. These mitochondria dysfunctions caused the defects in hearing organs. Strikingly, mtu1-/- mutant zebrafish displayed the abnormal startle response and swimming behaviors, significant decreases in the sizes of saccular otolith and numbers of hair cells in the auditory and vestibular organs. Furthermore, mtu1-/- mutant zebrafish exhibited the significant reductions in the hair bundle densities in utricle, saccule and lagena. Therefore, our findings may provide new insights into the pathophysiology of deafness, which was manifested by the deficient modifications at wobble position of mitochondrial tRNAs.