Biology subjects

Niwa, T.

Publications and source records attributed to Niwa, T..

4 recordsLinked to original sources

bioRxiv2021

Optimization of an LNP-mRNA vaccine candidate targeting SARS-CoV-2 receptor-binding domain

In 2020, two mRNA-based vaccines, encoding the full length of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, have been introduced for control of the coronavirus disease (COVID-19) pandemic1,2. However, reactogenicity, such as fever, caused by innate immune responses to the vaccine formulation remains to be improved. Here, we optimized a lipid nanoparticle (LNP)-based mRNA vaccine candidate, encoding the SARS-CoV-2 spike protein receptor-binding domain (LNP-mRNA-RBD), which showed improved immunogenicity by removing reactogenic materials from the vaccine formulation and protective potential against SARS-CoV-2 infection in cynomolgus macaques. LNP-mRNA-RBD induced robust antigen-specific B cells and follicular helper T cells in the BALB/c strain but not in the C57BL/6 strain; the two strains have contrasting abilities to induce type I interferon production by dendritic cells. Removal of reactogenic materials from original synthesized mRNA by HPLC reduced type I interferon (IFN) production by dendritic cells, which improved immunogenicity. Immunization of cynomolgus macaques with an LNP encapsulating HPLC-purified mRNA induced robust anti-RBD IgG in the plasma and in various mucosal areas, including airways, thereby conferring protection against SARS-CoV-2 infection. Therefore, fine-tuning the balance between the immunogenic and reactogenic activity of mRNA-based vaccine formulations may offer safer and more efficacious outcomes.

immunology↗

bioRxiv2021

Nascent polypeptide within the exit tunnel ensures continuous translation elongation by stabilizing the translating ribosome

Continuous translation elongation, irrespective of amino acid sequences, is a prerequisite for living organisms to produce their proteomes. However, the risk of elongation abortion is concealed within nascent polypeptide products. Negatively charged sequences with occasional intermittent prolines, termed intrinsic ribosome destabilization (IRD) sequences, destabilizes the translating ribosomal complex. Thus, some nascent chain sequences lead to premature translation cessation. Here, we show that the risk of IRD is maximal at the N-terminal regions of proteins encoded by dozens of Escherichia coli genes. In contrast, most potential IRD sequences in the middle of open reading frames remain cryptic. We found two elements in nascent chains that counteract IRD: the nascent polypeptide itself that spans the exit tunnel and its bulky amino acid residues that occupy the tunnel entrance region. Thus, nascent polypeptide products have a built-in ability to ensure elongation continuity by serving as a bridge and thus by protecting the large and small ribosomal subunits from dissociation.

biochemistry↗

bioRxiv2020

Defective Repression of OLE3::LUC 1 (DROL1) is specifically required for the splicing of AT-AC-type introns in Arabidopsis

An Arabidopsis mutant named defective repression of OLE3::LUC 1 (drol1) was originally isolated as a mutant with defects in the repression of OLEOSIN3 (OLE3) after seed germination. In this study, we show that DROL1 is an Arabidopsis homolog of yeast DIB1, a subunit of U5 snRNP in the spliceosome. It is also part of a new subfamily that is specific to a certain class of eukaryotes. Comprehensive analysis of the intron splicing using RNA-Seq analysis of the drol1 mutants revealed that most of the minor introns with AT-AC dinucleotide termini had reduced levels of splicing. Only two nucleotide substitutions from AT-AC to GT-AG enabled AT-AC-type introns to be spliced in drol1 mutants. Forty-eight genes, including those having important roles in abiotic stress responses and cell proliferation, exhibited reduced splicing of AT-AC-type introns in the drol1 mutants. Additionally, drol1 mutant seedlings showed retarded growth, similar to that caused by the activation of abscisic acid signaling, possibly as a result of reduced AT-AC-type intron splicing in the endosomal Na+/H+ antiporters and plant-specific histone deacetylases. These results indicate that DROL1 is specifically involved in the splicing of minor introns with AT-AC termini, and that this plays an important role in plant growth and development. Significance statementDefective Repression of OLE3::LUC 1 (DROL1) is a homolog of yeast DIB1, which is a subunit of the U5 snRNP in the spliceosome, but is also part of a new subfamily that is specific to a certain class of eukaryotes. Using RNA-Seq we show that introns with AT-AC dinucleotide termini were specifically retained in the transcriptome of drol1 mutants and that their splicing plays an important role in plant growth and development.

molecular biology↗

bioRxiv2020

Hierarchical Model for the Role of J-Domain Proteins in Distinct Cellular Functions

In Escherichia coli, the major bacterial Hsp70 system consists of DnaK, three J-domain proteins (JDPs: DnaJ, CbpA, and DjlA), and one nucleotide exchange factor (NEF: GrpE). JDPs determine substrate specificity for the Hsp70 system; however, knowledge on their specific role in bacterial cellular functions is limited. In this study, we demonstrated the role of JDPs in bacterial survival during heat stress and the DnaK-regulated formation of curli--extracellular amyloid fibers involved in E. coli biofilm formation. Genetic analysis with a complete set of JDP-null mutant strains demonstrated that only DnaJ is essential for survival at high temperature, while DnaJ and CbpA are indispensable in DnaK regulation of curli production. Additionally, we found that DnaJ and CbpA are involved in the expression of the master regulator CsgD through the folding of MlrA; this keeps CsgA in a translocation-competent state by preventing its aggregation in the cytoplasm. Our findings support a hierarchical model wherein the role of JDPs in the Hsp70 system differs according to individual cellular functions.

molecular biology↗