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Related Concept Videos

Pre-mRNA Processing: Modification of pre-mRNA Ends01:35

Pre-mRNA Processing: Modification of pre-mRNA Ends

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.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a cap to the 5' end of the growing transcript. In this process, a 5' phosphate is replaced by modified guanosine that has a methyl group attached (7-methyl guanosine). This 5' cap helps the cell...
pre-mRNA Processing02:01

pre-mRNA Processing

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.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it (7-Methyl guanosine). This 5’ cap helps the...
Pre-mRNA Processing02:01

Pre-mRNA Processing

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.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it (7-Methyl guanosine). This 5’ cap helps the...
Nonsense-mediated mRNA Decay02:27

Nonsense-mediated mRNA Decay

The Upf proteins that carry out nonsense-mediated decay (NMD) are found in all eukaryotic organisms, including humans. Each protein has an individual role, but they need to work in collaboration. Upf1 is an ATP-dependent RNA helicase that unwinds the RNA helix. Because Upf1 can unwind any RNA, Upf2 and Upf3 are required to help Upf1 discriminate between nonsense and normal mRNAs.
Usually, Upf3 binds to an Exon Junction Complex (EJC) at mRNA splice sites. If a ribosome fully translates the mRNA,...
Transfer RNA Synthesis02:36

Transfer RNA Synthesis

One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.
Each of these chemical modifications is carried by a specific enzyme, post-transcription. All of these enzymes have unique base and site-specificity. Methylation, the most common chemical modification, is carried by at least nine different enzymes, with...
RNA Editing02:23

RNA Editing

RNA editing is a post-transcriptional modification where a precursor mRNA (pre-mRNA) nucleotide sequence is changed by base insertion, deletion, or modification. The extent of RNA editing varies from a few hundred bases, in mitochondrial DNA of trypanosomes, to a just single base, in nuclear genes of mammals. Even a single base change in the pre-mRNA can convert a codon for one amino acid into the codon for another amino acid or a stop codon. This type of re-coding can significantly affect the...

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Related Experiment Video

Updated: Jun 13, 2026

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection
08:55

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

Published on: March 26, 2012

Thio-modification effects on mRNA translation using a PureCap-based capping method.

Zheyu Meng1, Yiwei Liu1, Yuko Nakashima1

  • 1Department of Chemistry, Graduate School of Science, Nagoya University Furo-cho, Chikusa-ku Nagoya 464-8602 Japan kimura.yasuaki.r9@f.mail.nagoya-u.ac.jp abe.hiroshi.p4@f.mail.nagoya-u.ac.jp.

RSC Chemical Biology
|June 12, 2026
PubMed
Summary
This summary is machine-generated.

Chemical modifications like 4'-thio and phosphorothioate (PS) substitutions on messenger RNA (mRNA) can enhance its therapeutic potential. These modifications, particularly at the 5' end, improve protein expression and stability for mRNA therapeutics.

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Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)
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Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

Published on: August 22, 2019

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Last Updated: Jun 13, 2026

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection
08:55

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

Published on: March 26, 2012

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)
11:46

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

Published on: August 22, 2019

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biotechnology

Background:

  • Chemical modifications are crucial for optimizing mRNA therapeutics, addressing challenges in translational efficiency, stability, and immune recognition.
  • Sugar (4 omino-thio) and phosphate-backbone (phosphorothioate, PS) modifications alter nucleic acid properties.
  • The PureCap method allows for the synthesis of highly pure capped mRNA for accurate assessment of modification effects.

Purpose of the Study:

  • To synthesize novel cap analogs with 4 omino-thio and PS substitutions at the +1 and +2 positions of mRNA.
  • To evaluate the impact of these modifications on mRNA production yields and protein expression.
  • To compare the efficacy of these modified mRNAs with existing mRNA capping strategies.

Main Methods:

  • Synthesis of cap analogs using the established PureCap method.
  • In vitro transcription (IVT) and HPLC purification of modified mRNAs.
  • Cell-based translation assays in HeLa cells and subcutaneous administration in mice to assess protein expression.

Main Results:

  • IVT yields were not significantly reduced by the introduction of 4 omino-thio or PS modifications.
  • PS-modified mRNAs showed higher protein expression than 4 omino-thio-modified mRNAs across Cap 0, Cap 1, and Cap 2 structures.
  • For Cap 2 structures, PureCap-derived 4 omino-thio and PS modifications yielded protein expression comparable to m 1 Ψ-modified mRNA (CleanCap AG) in both cell assays and mouse studies.

Conclusions:

  • 4 omino-thio and PS modifications at the 5 omino-terminal +1 and +2 nucleotides can modulate mRNA function.
  • The PureCap platform is versatile for developing mRNA therapeutics with enhanced properties.
  • These findings support the broad applicability of the PureCap platform in mRNA therapeutic development.