Analysis of enhanced protein production mediated by 5'-UTR intron

2019 Fiscal Year Final Research Report

• Project/Area Number: 17K08198
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Applied molecular and cellular biology
• Research Institution: Yamaguchi University
• Principal Investigator: HOSHIDA Hisashi 山口大学, 大学院創成科学研究科, 准教授 (00314823)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Keywords: イントロン / IME / スプライシング / Saccharomyces cerevisiae / ルシフェラーゼ / 最適化コドン

Outline of Final Research Achievements

The essential sequence in an intron for, a sequence in a expressed gene related to, and genes involved in intron-mediated enhancement (IME) were analyzed in Saccharomyces cerevisiae.
Most of mutations in the sequences conserved among introns in S. cerevisiae lost IME. These conserved sequences are essential for splicing. However, a mutation in the 3’ conserved sequence showed IME comparable to wild-type sequence. An RT-PCR analysis revealed that the 3’ mutant was not spliced. In addition, other several deletions in a non-conserved region showed IME but were not spliced. These results indicated that splicing is not necessary for IME. The genes involved in IME including basal repression of gene expression were screened using a knockout strain collection of S. cerevisiae. As a result, several genes were identified. Some of the genes affected mRNA accumulation but the others did not, indicating that the genes function in different steps of gene expression process.

Free Research Field

分子細胞生物学

Academic Significance and Societal Importance of the Research Achievements

本研究により，発現増強にとって必ずしもスプライシングが必要ないことを明らかにした。また，発現増強機能の解明には関わる遺伝子を同定しそれら遺伝子の機能を明らかにすることが必要であり，本研究で発現増強および基本的な発現抑制に関与する遺伝を同定したことで，IMEのメカニズムの一端が明らかになり，さらなる解明を進めることが可能になった。
現在，人工設計した合成遺伝子を用いた物質生産細胞の構築が盛んになってきている。遺伝子設計において，設計遺伝子が発現することは必須である。本研究で明らかにした発現抑制配列は，その完全な理解には至っていないものの，今後の遺伝子設計にとっての重要な知見である。