Graphical Abstract Highlights d Tumorigenesis depends on functional OXPHOS d OXPHOS-derived ATP is not required for tumor formation d DHODH-driven pyrimidine biosynthesis requires CoQ redoxcycling d CoQ redox-cycling via OXPHOS drives tumorigenesis through pyrimidine biosynthesis

The de novo design of amino acid sequences to fold into desired structures is a way to reach a more thorough understanding of how amino acid sequences encode protein structures and to supply methods for protein engineering. Notwithstanding significant breakthroughs, there are noteworthy limitations in current computational protein design. To overcome them needs computational models to complement current ones and experimental tools to provide extensive feedbacks to theory. Here we develop a comprehensive statistical energy function for protein design with a new general strategy and verify that it can complement and rival current well-established models. We establish that an experimental approach can be used to efficiently assess or improve the foldability of designed proteins. We report four de novo proteins for different targets, all experimentally verified to be well-folded, solved solution structures for two being in excellent agreement with respective design targets.

Motivation The recent discovery of numerous non-coding RNAs (long non-coding RNAs, in particular) has transformed our perception about the roles of RNAs in living organisms. Our ability to understand them, however, is hampered by our inability to solve their secondary and tertiary structures in high resolution efficiently by existing experimental techniques. Computational prediction of RNA secondary structure, on the other hand, has received much-needed improvement, recently, through deep learning of a large approximate data, followed by transfer learning with gold-standard base-pairing structures from high-resolution 3-D structures. Here, we expand this single-sequence-based learning to the use of evolutionary profiles and mutational coupling. Results The new method allows large improvement not only in canonical base-pairs (RNA secondary structures) but more so in base-pairing associated with tertiary interactions such as pseudoknots, noncanonical and lone base-pairs. In particular, it is highly accurate for those RNAs of more than 1000 homologous sequences by achieving >0.8 F1-score (harmonic mean of sensitivity and precision) for 14/16 RNAs tested. The method can also significantly improve base-pairing prediction by incorporating artificial but functional homologous sequences generated from deep mutational scanning without any modification. The fully automatic method (publicly available as server and standalone software) should provide the scientific community a new powerful tool to capture not only the secondary structure but also tertiary base-pairing information for building three-dimensional models. It also highlights the future of accurately solving the base-pairing structure by using a large number of natural and/or artificial homologous sequences. Availability Standalone-version of SPOT-RNA2 is available at https://github.com/jaswindersingh2/SPOT-RNA2. Direct prediction can also be made at https://sparks-lab.org/server/spot-rna2/. The datasets used in this research can also be downloaded from the GITHUB and the webserver mentioned above.

Despite the large number of noncoding RNAs in human genome and their roles in many diseases include cancer, we know very little about them due to lack of structural clues. The centerpiece of the structural clues is the full RNA base-pairing structure of secondary and tertiary contacts that can be precisely obtained only from costly and time-consuming 3D structure determination. Here, we performed deep mutational scanning of self-cleaving CPEB3 ribozyme by error-prone PCR and showed that a library of <5 × 104 single-to-triple mutants is sufficient to infer 25 of 26 base pairs including non-nested, nonhelical, and noncanonical base pairs with both sensitivity and precision at 96%. Such accurate inference was further confirmed by a twister ribozyme at 100% precision with only noncanonical base pairs as false negatives. The performance was resulted from analyzing covariation-induced deviation of activity by utilizing both functional and nonfunctional variants for unsupervised classification, followed by Monte Carlo (MC) simulated annealing with mutation-derived scores. Highly accurate inference can also be obtained by combining MC with evolution/direct coupling analysis, R-scape or epistasis analysis. The results highlight the usefulness of deep mutational scanning for high-accuracy structural inference of self-cleaving ribozymes with implications for other structured RNAs that permit high-throughput functional selections.

Motivation The accuracy of RNA secondary and tertiary structure prediction can be significantly improved by using structural restraints derived from evolutionary coupling or direct coupling analysis. Currently, these coupling analyses relied on manually curated multiple sequence alignments collected in the Rfam database, which contains 3016 families. By comparison, millions of non-coding RNA sequences are known. Here, we established RNAcmap, a fully automatic pipeline that enables evolutionary coupling analysis for any RNA sequences. The homology search was based on the covariance model built by INFERNAL according to two secondary structure predictors: a folding-based algorithm RNAfold and the latest deep-learning method SPOT-RNA. Results We showed that the performance of RNAcmap is less dependent on the specific evolutionary coupling tool but is more dependent on the accuracy of secondary structure predictor with the best performance given by RNAcmap (SPOT-RNA). The performance of RNAcmap (SPOT-RNA) is comparable to that based on Rfam-supplied alignment and consistent for those sequences that are not in Rfam collections. Further improvement can be made with a simple meta predictor RNAcmap (SPOT-RNA/RNAfold) depending on which secondary structure predictor can find more homologous sequences. Reliable base-pairing information generated from RNAcmap, for RNAs with high effective homologous sequences, in particular, will be useful for aiding RNA structure prediction. Availability RNAcmap is available as a web server at https://sparks-lab.org/server/rnacmap/ and as a standalone application along with the datasets at https://github.com/sparks-lab-org/RNAcmap_standalone. A platform independent and fully configured docker image of RNAcmap is also provided at https://hub.docker.com/r/jaswindersingh2/rnacmap.