Takayuki Katoh 교수

The steady-state levels of microRNAs (miRNAs) and their activities are regulated by the post-transcriptional processes. It is known that 3' ends of several miRNAs undergo post-dicing adenylation or uridylation. We isolated the liver-specific miR-122 from human hepatocytes and mouse livers. Direct analysis by mass spectrometry revealed that one variant of miR-122 has a 3'-terminal adenosine that is introduced after processing by Dicer. We identified GLD-2, which is a regulatory cytoplasmic poly(A

A bacterial translation factor EF-P alleviates ribosomal stalling caused by polyproline sequence by accelerating Pro-Pro formation. EF-P recognizes a specific D-arm motif found in tRNAPro isoacceptors, 9-nt D-loop closed by a stable D-stem sequence, for Pro-selective peptidyl-transfer acceleration. It is also known that the T-stem sequence on aminoacyl-tRNAs modulates strength of the interaction with EF-Tu, giving enhanced incorporation of non-proteinogenic amino acids such as some N-methyl amin

Due to their unique characteristics, which are not shared by canonical α-peptides, peptides that contain stretches of consecutive β-amino acids are attractive scaffolds for novel peptide drugs and nanomaterials. Although ribosomal incorporation of single or nonconsecutive β-amino acids into peptides has previously been reported, the incorporation of consecutive β-amino acids has not yet been accomplished. This is primarily due to their incompatibility with the ribosomal translation system. Here,

Small interfering RNA (siRNA) induces sequence-specific post-transcriptional gene silencing in mammalian cells. Different efficacy of each siRNA is considered to result from sequence preference by protein components in RNAi. To obtain mechanistic insight into siRNA functionality, here we describe a complete data set of siRNA activities targeting all possible position of a single mRNA in human cells. Seven hundred and two siRNAs covering open reading frame of enhanced green fluorescent protein mR

The ribosome stalls on translation of polyproline sequences due to inefficient peptide bond formation between consecutive prolines. The translation factor EF-P is able to alleviate this stalling by accelerating Pro-Pro formation. However, the mechanism by which EF-P recognizes the stalled complexes and accelerates peptide bond formation is not known. Here, we use genetic code reprogramming through a flexible in-vitro translation (FIT) system to investigate how mutations in tRNA(Pro) affect EF-P

MicroRNA-122 (miR-122), which is expressed at high levels in hepatocytes, is selectively stabilized by 3'-adenylation mediated by the cytoplasmic poly(A) polymerase GLD-2. Here, we report that poly(A)-specific ribonuclease (PARN) is responsible for the deadenylation and destabilization of miR-122. The 3'-oligoadenylated variant of miR-122 was detected in Huh7 cells when PARN was down-regulated. In addition, both the steady-state level and stability of miR-122 were increased in PARN knockdown cel

Because γ-amino acids generally undergo rapid self-cyclization upon esterification on the carboxyl group, for example, γ-aminoacyl-tRNA, there are no reports of the ribosomal elongation of γ-amino acids to the best of our knowledge. To avoid such self-cyclization, we utilized cyclic γ-amino acids and demonstrated their elongation into a peptide chain. Although the incorporation of the cyclic γ-amino acids is intrinsically slow, we here show that the combination of elongation factor P and enginee

2-Aminobenzoic acid and heterocyclic amino acids are aromatic homologues of β-amino acids, but their chemical properties are quite distinct. Because of the poor nucleophilicity of the amino group conjugated with the aromatic ring, no literature reports of their successful ribosomal elongation in nascent peptide chains have appeared to date. Here we report for the first time their incorporation in nascent peptide chains by means of a reconstituted translation system in which a designer tRNA<sup>P

d/l-Hybrid peptides are an attractive class of molecular modality because they are able to exhibit high proteolytic stability and unique structural diversity which cannot be accessed by those consisting of only proteinogenic l-amino acids. Despite such an expectation, it has not been possible to devise de novo d/l-hybrid peptides capable of disrupting the function of a protein target(s) due to the lack of an effective method that reliably constructs a highly diverse library and screens active sp

Genetic code reprogramming has enabled us to ribosomally incorporate various nonproteinogenic amino acids (npAAs) into peptides in vitro. The repertoire of usable npAAs has been expanded to include not only l-α-amino acids with noncanonical sidechains but also those with noncanonical backbones. Despite successful single incorporation of npAAs, multiple and consecutive incorporations often suffer from low efficiency or are even unsuccessful. To overcome this stumbling block, engineering approache

A wealth of knowledge has been accumulated on ribosomal synthesis of macrocyclic peptides in the past decade. In nature, backbone cyclization of the translated linear peptides is generally catalyzed by specific enzymes, giving them peptidase resistance, thermodynamic stability and various other physiological activities. Due to these biochemical traits, backbone cyclic peptides have become an attractive resource for the discovery of drug leads. Recently, various new methodologies have also been e

α-Aminoxy and α-hydrazino acids are β-amino acid analogs with β-carbons replaced by oxygen and nitrogen, respectively. Such heteroatoms dictate the folding of peptides into specific secondary structures called pseudo-γ-turns. Achiral α-aminoxyacetic acid (^NOGly) and l-α-hydrazinophenylalanine (l-^NNPhe) have been shown to be suitable for single incorporation during ribosomal translation, but whether ribosomes tolerate other types of α-aminoxy/α-hydrazino acids with l/d-confi

Aromatic cyclic β^2,3-amino acids (cβAAs), such as 2-aminobenzoic acid and 3-aminothiophene-2-carboxylic acid, are building blocks that can induce unique folding propensities of peptides. Although their ribosomal elongation had been a formidable task due to the low nucleophilicity of their amino groups, we have recently overcome this issue by means of an engineered tRNA^Pro1E2 that enhances their incorporation efficiency into nascent peptide chains. Here we report ribosomal s