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During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA.  Marilyn Kozak discovered that the sequence RCCAUGG (where R...
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Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...
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Alternative RNA splicing is the regulated splicing of exons and introns to produce different mature mRNAs from a single pre-mRNA. Unlike in constitutive splicing where a single gene produces a single type of mRNA, alternative splicing allows an organism to produce multiple proteins from a single gene and plays an important role in protein diversity.
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In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA to distinguish it from mature mRNA.
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Using the E1A Minigene Tool to Study mRNA Splicing Changes
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Improved protein splicing through viral passaging.

Adam J Hume1,2,3, Dylan J Deeney1,2, John S Smetana4

  • 1Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts, USA.

Mbio
|May 23, 2024
PubMed
Summary
This summary is machine-generated.

Serial passaging of a recombinant Ebola virus engineered with an intervening protein (intein) reporter led to mutations that enhance intein splicing activity. These intein mutations improve catalytic efficiency across different systems and contexts, offering a new method for intein development.

Keywords:
Ebola virusinteinprotein splicingreporter virusself-splicing fluorescent reporter

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Area of Science:

  • Molecular Biology
  • Virology
  • Protein Engineering

Background:

  • Intervening proteins (inteins) are protein elements that excise themselves and ligate flanking sequences (exteins).
  • Inteins have been engineered into reporters for biological systems, such as a self-removing translation reporter for Ebola virus (EBOV) using the RadA intein and ZsGreen fluorescent protein (ZsG).
  • Previous incorporation of this reporter into recombinant EBOV (rEBOV-RadA-ZsG) resulted in a replication defect, potentially due to insufficient RadA splicing.

Purpose of the Study:

  • To investigate the effect of serial passaging on the replication efficiency of rEBOV-RadA-ZsG.
  • To identify and characterize mutations that enhance intein activity within a viral context.
  • To establish a novel strategy for selecting inteins with improved catalytic properties.

Main Methods:

  • Serial passaging of rEBOV-RadA-ZsG in human cells.
  • Sequencing of passaged viral genomes to identify mutations.
  • Assessing intein activity in both prokaryotic and eukaryotic expression systems with various extein contexts.

Main Results:

  • Serial passaging significantly increased the replication efficiency of rEBOV-RadA-ZsG compared to unpassaged virus.
  • Sequencing revealed specific intein-associated mutations.
  • These identified mutations enhanced intein activity across diverse expression systems and extein environments.

Conclusions:

  • Viral passaging is an effective method for isolating inteins with enhanced catalytic activity.
  • The identified mutations confer improved intein splicing efficiency, independent of extein context or expression system.
  • This work presents a new approach for developing highly active inteins for protein engineering applications.