Development of PCR primers enabling the design of flexible sticky ends for efficient concatenation of long DNA fragments.

We developed chemically modified PCR primers that allow the design of flexible sticky ends by introducing a photo-cleavable group at the phosphate moiety. Nucleic acid derivatives containing o-nitrobenzyl photo-cleavable groups with a tert-butyl group at the benzyl position were stable during strong base treatment for oligonucleotide synthesis and thermal cycling in PCR reactions. PCR using primers incorporating these nucleic acid derivatives confirmed that chain extension reactions completely stopped at position 1 before and after the site of the photo-cleavable group was introduced. DNA fragments of 2 and 3 kbp, with sticky ends of 50 bases, were successfully concatenated with a high yield of 77%. A plasmid was constructed using this method. Finally, we applied this approach to construct a 48.5 kbp lambda phage DNA, which is difficult to achieve using restriction enzyme-based methods. After 7 days, we were able to confirm the generation of DNA of the desired length. Although the efficiency is yet to be improved, the chemically modified PCR primer offers potential to complement enzymatic methods and serve as a DNA concatenation technique.

The Effect of γ Phosphate Modified Deoxynucleotide Substrates on PCR Activity and Fidelity.

Controlling PCR fidelity is an important issue for molecular biology and high-fidelity PCR is essential for gene cloning. In general, fidelity control is achieved by protein engineering of polymerases. In contrast, only a few studies have reported controlling fidelity using chemically modified nucleotide substrates. In this report, we synthesized nucleotide substrates possessing a modification on Pγ and evaluated the effect of this modification on PCR fidelity. One of the substrates, nucleotide tetraphosphate, caused a modest decrease in Taq DNA polymerase activity and the effect on PCR fidelity was dependent on the type of mutation. The use of deoxyadenosine tetraphosphate enhanced the A : T→G : C mutation dramatically, which is common when using Taq polymerase. Conversely, deoxyguanosine tetraphosphate (dG4P) suppressed this mutation but increased the G : C→A : T mutation during PCR. Using an excess amount of dG4P suppressed both mutations successfully and total fidelity was improved.

Remarkable improvement in detection of readthrough downstream-of-gene transcripts by semi-extractable RNA-sequencing.

The mammalian cell nucleus contains dozens of membrane-less nuclear bodies that play significant roles in various aspects of gene expression. Several nuclear bodies are nucleated by specific architectural noncoding RNAs (arcRNAs) acting as structural scaffolds. We have reported that a minor population of cellular RNAs exhibits an unusual semi-extractable feature upon using the conventional procedure of RNA preparation and that needle shearing or heating of cell lysates remarkably improves extraction of dozens of RNAs. Because semi-extractable RNAs, including known arcRNAs, commonly localize in nuclear bodies, this feature may be a hallmark of arcRNAs. Using the semi-extractability of RNA, we performed genome-wide screening of semi-extractable long noncoding RNAs to identify new candidate arcRNAs for arcRNA under hyperosmotic and heat stress conditions. After screening stress-inducible and semi-extractable RNAs, hundreds of readthrough downstream-of-gene (DoG) transcripts over several hundreds of kilobases, many of which were not detected among RNAs prepared by the conventional extraction procedure, were found to be stress-inducible and semi-extractable. We further characterized some of the abundant DoGs and found that stress-inducible transient extension of the 3'-UTR made DoGs semi-extractable. Furthermore, they were localized in distinct nuclear foci that were sensitive to 1,6-hexanediol. These data suggest that semi-extractable DoGs exhibit arcRNA-like features and our semi-extractable RNA-seq is a powerful tool to extensively monitor DoGs that are induced under specific physiological conditions.

A streamlined strain engineering workflow with genome-wide screening detects enhanced protein secretion in Komagataella phaffii.

Expression of secreted recombinant proteins burdens the protein secretion machinery, limiting production. Here, we describe an approach to improving protein production by the non-conventional yeast Komagataella phaffii comprised of genome-wide screening for effective gene disruptions, combining them in a single strain, and recovering growth reduction by adaptive evolution. For the screen, we designed a multiwell-formatted, streamlined workflow to high-throughput assay of secretion of a single-chain small antibody, which is cumbersome to detect but serves as a good model of proteins that are difficult to secrete. Using the consolidated screening system, we evaluated >19,000 mutant strains from a mutant library prepared by a modified random gene-disruption method, and identified six factors for which disruption led to increased antibody production. We then combined the disruptions, up to quadruple gene knockouts, which appeared to contribute independently, in a single strain and observed an additive effect. Target protein and promoter were basically interchangeable for the effects of knockout genes screened. We finally used adaptive evolution to recover reduced cell growth by multiple gene knockouts and examine the possibility for further enhancing protein secretion. Our successful, three-part approach holds promise as a method for improving protein production by non-conventional microorganisms.

Avoiding entry into intracellular protein degradation pathways by signal mutations increases protein secretion in Pichia pastoris.

In our previous study, we serendipitously discovered that protein secretion in the methylotrophic yeast Pichia pastoris is enhanced by a mutation (V50A) in the mating factor alpha (MFα) prepro-leader signal derived from Saccharomyces cerevisiae. In the present study, we investigated 20 single-amino-acid substitutions, including V50A, located within the MFα signal peptide, indicating that V50A and several single mutations alone provided significant increase in production of the secreted proteins. In addition to hydrophobicity index analysis, both an unfolded protein response (UPR) biosensor analysis and a microscopic observation showed a clear difference on the levels of UPR induction and mis-sorting of secretory protein into vacuoles among the wild-type and mutated MFα signal peptides. This work demonstrates the importance of avoiding entry of secretory proteins into the intracellular protein degradation pathways, an observation that is expected to contribute to the engineering of strains with increased production of recombinant secreted proteins.

QRNAstruct: a method for extracting secondary structural features of RNA via regression with biological activity.

Recent technological advances have enabled the generation of large amounts of data consisting of RNA sequences and their functional activity. Here, we propose a method for extracting secondary structure features that affect the functional activity of RNA from sequence-activity data. Given pairs of RNA sequences and their corresponding bioactivity values, our method calculates position-specific structural features of the input RNA sequences, considering every possible secondary structure of each RNA. A Ridge regression model is trained using the structural features as feature vectors and the bioactivity values as response variables. Optimized model parameters indicate how secondary structure features affect bioactivity. We used our method to extract intramolecular structural features of bacterial translation initiation sites and self-cleaving ribozymes, and the intermolecular features between rRNAs and Shine-Dalgarno sequences and between U1 RNAs and splicing sites. We not only identified known structural features but also revealed more detailed insights into structure-activity relationships than previously reported. Importantly, the datasets we analyzed here were obtained from different experimental systems and differed in size, sequence length and similarity, and number of RNA molecules involved, demonstrating that our method is applicable to various types of data consisting of RNA sequences and bioactivity values.

m6 A modification of HSATIII lncRNAs regulates temperature-dependent splicing.

Nuclear stress bodies (nSBs) are nuclear membraneless organelles formed around stress-inducible HSATIII architectural long noncoding RNAs (lncRNAs). nSBs repress splicing of hundreds of introns during thermal stress recovery, which are partly regulated by CLK1 kinase phosphorylation of temperature-dependent Ser/Arg-rich splicing factors (SRSFs). Here, we report a distinct mechanism for this splicing repression through protein sequestration by nSBs. Comprehensive identification of RNA-binding proteins revealed HSATIII association with proteins related to N6 -methyladenosine (m6 A) RNA modification. 11% of the first adenosine in the repetitive HSATIII sequence were m6 A-modified. nSBs sequester the m6 A writer complex to methylate HSATIII, leading to subsequent sequestration of the nuclear m6 A reader, YTHDC1. Sequestration of these factors from the nucleoplasm represses m6 A modification of pre-mRNAs, leading to repression of m6 A-dependent splicing during stress recovery phase. Thus, nSBs serve as a common platform for regulation of temperature-dependent splicing through dual mechanisms employing two distinct ribonucleoprotein modules with partially m6 A-modified architectural lncRNAs.

Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression.

In the yeast Saccharomyces cerevisiae, terminator sequences not only terminate transcription but also affect expression levels of the protein-encoded upstream of the terminator. The non-conventional yeast Pichia pastoris (syn. Komagataella phaffii) has frequently been used as a platform for metabolic engineering but knowledge regarding P. pastoris terminators is limited. To explore terminator sequences available to tune protein expression levels in P. pastoris, we created a 'terminator catalog' by testing 72 sequences, including terminators from S. cerevisiae or P. pastoris and synthetic terminators. Altogether, we found that the terminators have a tunable range of 17-fold. We also found that S. cerevisiae terminator sequences maintain function when transferred to P. pastoris. Successful tuning of protein expression levels was shown not only for the reporter gene used to define the catalog but also using betaxanthin production as an example application in pathway flux regulation. Moreover, we found experimental evidence that protein expression levels result from mRNA abundance and in silico evidence that levels reflect the stability of mRNA 3'-UTR secondary structure. In combination with promoter selection, the novel terminator catalog constitutes a basic toolbox for tuning protein expression levels in metabolic engineering and synthetic biology in P. pastoris.

Finding the direct optimal RNA barrier energy and improving pathways with an arbitrary energy model.

MOTIVATION: RNA folding kinetics plays an important role in the biological functions of RNA molecules. An important goal in the investigation of the kinetic behavior of RNAs is to find the folding pathway with the lowest energy barrier. For this purpose, most of the existing methods use heuristics because the number of possible pathways is huge even if only the shortest (direct) folding pathways are considered. RESULTS: In this study, we propose a new method using a best-first search strategy to efficiently compute the exact solution of the minimum barrier energy of direct pathways. Using our method, we can find the exact direct pathways within a Hamming distance of 20, whereas the previous methods even miss the exact short pathways. Moreover, our method can be used to improve the pathways found by existing methods for exploring indirect pathways. AVAILABILITY AND IMPLEMENTATION: The source code and datasets created and used in this research are available at https://github.com/eukaryo/czno. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Improving the prediction accuracy of protein abundance in Escherichia coli using mRNA accessibility.

RNA secondary structure around translation initiation sites strongly affects the abundance of expressed proteins in Escherichia coli. However, detailed secondary structural features governing protein abundance remain elusive. Recent advances in high-throughput DNA synthesis and experimental systems enable us to obtain large amounts of data. Here, we evaluated six types of structural features using two large-scale datasets. We found that accessibility, which is the probability that a given region around the start codon has no base-paired nucleotides, showed the highest correlation with protein abundance in both datasets. Accessibility showed a significantly higher correlation (Spearman's ρ = 0.709) than the widely used minimum free energy (0.554) in one of the datasets. Interestingly, accessibility showed the highest correlation only when it was calculated by a log-linear model, indicating that the RNA structural model and how to utilize it are important. Furthermore, by combining the accessibility and activity of the Shine-Dalgarno sequence, we devised a method for predicting protein abundance more accurately than existing methods. We inferred that the log-linear model has a broader probabilistic distribution than the widely used Turner energy model, which contributed to more accurate quantification of ribosome accessibility to translation initiation sites.

Promoter tools for further development of Aspergillus oryzae as a platform for fungal secondary metabolite production.

BACKGROUND: The filamentous fungus Aspergillus oryzae is widely used for secondary metabolite production by heterologous expression; thus, a wide variety of promoter tools is necessary to broaden the application of this species. Here we built a procedure to survey A. flavus genes constitutively highly expressed in 83 transcriptome datasets obtained under various conditions affecting secondary metabolite production, to find promoters useful for heterologous expression of genes in A. oryzae. RESULTS: To test the ability of the promoters of the top 6 genes to induce production of a fungal secondary metabolite, ustiloxin B, we inserted the promoters before the start codon of ustR, which encodes the transcription factor of the gene cluster responsible for ustiloxin B biosynthesis, in A. oryzae. Four of the 6 promoters induced ustiloxin B production in all tested media (solid maize, liquid V8 and PDB media), and also ustR expression. Two of the 4 promoters were those of tef1 and gpdA, which are well characterized in A. oryzae and A. nidulans, respectively, whereas the other two, those of AFLA_030930 and AFLA_113120, are newly reported here and show activities comparable to that of the gpdA promoter with respect to induction of gene expression and ustiloxin B production. CONCLUSION: We newly reported two sequences as promoter tools for secondary metabolite production in A. oryzae. Our results demonstrate that our simple strategy of surveying for constitutively highly expressed genes in large-scale transcriptome datasets is useful for finding promoter sequences that can be used as heterologous expression tools in A. oryzae.

LncRNA-dependent nuclear stress bodies promote intron retention through SR protein phosphorylation.

A number of long noncoding RNAs (lncRNAs) are induced in response to specific stresses to construct membrane-less nuclear bodies; however, their function remains poorly understood. Here, we report the role of nuclear stress bodies (nSBs) formed on highly repetitive satellite III (HSATIII) lncRNAs derived from primate-specific satellite III repeats upon thermal stress exposure. A transcriptomic analysis revealed that depletion of HSATIII lncRNAs, resulting in elimination of nSBs, promoted splicing of 533 retained introns during thermal stress recovery. A HSATIII-Comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) analysis identified multiple splicing factors in nSBs, including serine and arginine-rich pre-mRNA splicing factors (SRSFs), the phosphorylation states of which affect splicing patterns. SRSFs are rapidly de-phosphorylated upon thermal stress exposure. During stress recovery, CDC like kinase 1 (CLK1) was recruited to nSBs and accelerated the re-phosphorylation of SRSF9, thereby promoting target intron retention. Our findings suggest that HSATIII-dependent nSBs serve as a conditional platform for phosphorylation of SRSFs by CLK1 to promote the rapid adaptation of gene expression through intron retention following thermal stress exposure.

A robust model for quantitative prediction of the silencing efficacy of wild-type and A-to-I edited miRNAs.

MicroRNAs (miRNAs) are small non-coding RNAs that play essential roles in the regulation of gene function by a mechanism known as RNA silencing. In a previous study, we revealed that miRNA-mediated silencing efficacy is correlated with the combinatorial thermodynamic properties of the miRNA seed-target mRNA duplex and the 5´-terminus of the miRNA duplex, which can be predicted using 'miScore'. In this study, a robust refined-miScore was developed by integrating the thermodynamic properties of various miRNA secondary structures and the latest thermodynamic parameters of wobble base-pairing, including newly established parameters for I:C base pairing. Through repeated random sampling and machine learning, refined-miScore models calculated with either melting temperature (Tm) or free energy change (ΔG) values were successfully built and validated in both wild-type and adenosine-to-inosine edited miRNAs. In addition to the previously reported contribution of the seed-target duplex and 5´-terminus region, the refined-miScore suggests that the central and 3´-terminus regions of the miRNA duplex also play a role in the thermodynamic regulation of miRNA-mediated silencing efficacy.

Computational prediction of lncRNA-mRNA interactionsby integrating tissue specificity in human transcriptome.

Long noncoding RNAs (lncRNAs) play a key role in normal tissue differentiation and cancer development through their tissue-specific expression in the human transcriptome. Recent investigations of macromolecular interactions have shown that tissue-specific lncRNAs form base-pairing interactions with various mRNAs associated with tissue-differentiation, suggesting that tissue specificity is an important factor controlling human lncRNA-mRNA interactions.Here, we report investigations of the tissue specificities of lncRNAs and mRNAs by using RNA-seq data across various human tissues as well as computational predictions of tissue-specific lncRNA-mRNA interactions inferred by integrating the tissue specificity of lncRNAs and mRNAs into our comprehensive prediction of human lncRNA-RNA interactions. Our predicted lncRNA-mRNA interactions were evaluated by comparisons with experimentally validated lncRNA-mRNA interactions (between the TINCR lncRNA and mRNAs), showing the improvement of prediction accuracy over previous prediction methods that did not account for tissue specificities of lncRNAs and mRNAs. In addition, our predictions suggest that the potential functions of TINCR lncRNA not only for epidermal differentiation but also for esophageal development through lncRNA-mRNA interactions. REVIEWERS: This article was reviewed by Dr. Weixiong Zhang and Dr. Bojan Zagrovic.

Evolutionary design of multiple genes encoding the same protein.

MOTIVATION: Enhancing expression levels of a target protein is an important goal in synthetic biology. A widely used strategy is to integrate multiple copies of genes encoding a target protein into a host organism genome. Integrating highly similar sequences, however, can induce homologous recombination between them, resulting in the ultimate reduction of the number of integrated genes. RESULTS: We propose a method for designing multiple protein-coding sequences (i.e. CDSs) that are unlikely to induce homologous recombination, while encoding the same protein. The method, which is based on multi-objective genetic algorithm, is intended to design a set of CDSs whose nucleotide sequences are as different as possible and whose codon usage frequencies are as highly adapted as possible to the host organism. We show that our method not only successfully designs a set of intended CDSs, but also provides insight into the trade-off between nucleotide differences among gene copies and codon usage frequencies. AVAILABILITY AND IMPLEMENTATION: Our method, named Tandem Designer, is available as a web-based application at http://tandem.trahed.jp/tandem/ . CONTACT: : terai_goro@intec.co.jp or asai@k.u-tokyo.ac.jp. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Identification of a putative FR901469 biosynthesis gene cluster in fungal sp. No. 11243 and enhancement of the productivity by overexpressing the transcription factor gene frbF.

FR901469 is an antifungal antibiotic produced by fungal sp. No. 11243. Here, we searched for FR901469 biosynthesis genes in the genome of No. 11243. Based on the molecular structure of FR901469 and endogenous functional motifs predicted in each genomic NRPS gene, a putative FR901469 biosynthesis gene cluster harboring the most plausible NRPS gene was identified. A transcription factor gene, designated frbF, was found in the cluster. To improve FR901469 productivity, we constructed a strain in which frbF was overexpressed and named it TFH2-2. FR901469 productivity of TFH2-2 was 3.4 times higher than that of the wild-type strain. Transcriptome analysis revealed that most of the genes in the putative FR901469 biosynthesis gene cluster were upregulated in TFH2-2. It also showed that the expression of genes related to ergosterol biosynthesis, ß-1,3-glucan catabolism, and chitin synthesis was inclined to exhibit significant differences in TFH2-2.

Genome Sequence of Ustilaginoidea virens IPU010, a Rice Pathogenic Fungus Causing False Smut.

Ustilaginoidea virens is a rice pathogenic fungus that causes false smut disease, a disease that seriously damages the yield and quality of the grain. Analysis of the U. virens IPU010 33.6-Mb genome sequence will aid in the understanding of the pathogenicity of the strain, particularly in regard to effector proteins and secondary metabolic genes.

Comprehensive prediction of lncRNA-RNA interactions in human transcriptome.

MOTIVATION: Recent studies have revealed that large numbers of non-coding RNAs are transcribed in humans, but only a few of them have been identified with their functions. Identification of the interaction target RNAs of the non-coding RNAs is an important step in predicting their functions. The current experimental methods to identify RNA-RNA interactions, however, are not fast enough to apply to a whole human transcriptome. Therefore, computational predictions of RNA-RNA interactions are desirable, but this is a challenging task due to the huge computational costs involved. RESULTS: Here, we report comprehensive predictions of the interaction targets of lncRNAs in a whole human transcriptome for the first time. To achieve this, we developed an integrated pipeline for predicting RNA-RNA interactions on the K computer, which is one of the fastest super-computers in the world. Comparisons with experimentally-validated lncRNA-RNA interactions support the quality of the predictions. Additionally, we have developed a database that catalogs the predicted lncRNA-RNA interactions to provide fundamental information about the targets of lncRNAs.

CDSfold: an algorithm for designing a protein-coding sequence with the most stable secondary structure.

MOTIVATION: An important problem in synthetic biology is to design a nucleotide sequence of an mRNA that confers a desirable expression level of a target protein. The secondary structure of protein-coding sequences (CDSs) is one potential factor that could have both positive and negative effects on protein production. To elucidate the role of secondary structure in CDSs, algorithms for manipulating secondary structure should be developed. RESULTS: We developed an algorithm for designing a CDS with the most stable secondary structure among all possible ones translated into the same protein, and implemented it as the program CDSfold. The algorithm runs the Zuker algorithm under the constraint of a given amino acid sequence. The time and space complexity is O(L(3)) and O(L(2)), respectively, where L is the length of the CDS to be designed. Although our algorithm is slower than the original Zuker algorithm, it could design a relatively long (2.7-kb) CDS in approximately 1 h. AVAILABILITY AND IMPLEMENTATION: The CDSfold program is freely available for non-commercial users as stand-alone and web-based software from http://cdsfold.trahed.jp/cdsfold/ CONTACTS: terai-goro@aist.go.jp or asai@k.u-tokyo.ac.jp SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genome Sequence of Fungal Species No.11243, Which Produces the Antifungal Antibiotic FR901469.

Fungal species No.11243 was originally isolated from a decayed leaf sample collected in Kyoto, Japan. It produces FR901469, a 1,3-beta-glucan synthase inhibitor. The genome sequence of No.11243 was determined and annotated to obtain useful information for improving productivity of the effective antifungal agent FR901469.