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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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Initiating translation is complex because it involves multiple molecules. Initiator tRNA, ribosomal subunits, and eukaryotic initiation factors (eIFs) are all required to assemble on the initiation codon of mRNA. This process consists of several steps that are mediated by different eIFs.
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Translation01:31

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Translation is the process of synthesizing proteins from the genetic information carried by messenger RNA (mRNA). Following transcription, it constitutes the final step in the expression of genes. This process is carried out by ribosomes, complexes of protein and specialized RNA molecules. Ribosomes, transfer RNA (tRNA), and other proteins produce a chain of amino acids—the polypeptide—as the end product of translation.
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Nonsense-mediated mRNA Decay02:27

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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.
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Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
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Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific...
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Cryptic intronic transcriptional initiation generates efficient endogenous mRNA templates for C9orf72-associated RAN

Shannon L Miller1,2, Katelyn M Green1,2, Bradley Crone3

  • 1Department of Neurology, University of Michigan, Ann Arbor, MI 48109.

Proceedings of the National Academy of Sciences of the United States of America
|August 4, 2025
PubMed
Summary

Intronic repeat expansions in C9orf72 cause ALS and FTD. New research identifies cryptic, intron-initiated C9orf72 mRNAs as the main source for toxic dipeptide repeat protein production via repeat-associated non-AUG translation.

Keywords:
ALSneurodegenerationrepeat expansion diseasetranscriptiontranslation

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Area of Science:

  • Neuroscience
  • Genetics
  • Molecular Biology

Background:

  • C9orf72 repeat expansions are the leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
  • Pathogenic dipeptide repeat (DPR) proteins are produced through repeat-associated non-AUG (RAN) translation, but the endogenous RNA template remains unidentified.
  • Understanding the source of these toxic proteins is crucial for developing effective therapies.

Purpose of the Study:

  • To identify the endogenous RNA template responsible for DPR protein synthesis in C9orf72-associated neurodegenerative diseases.
  • To investigate the role of different C9orf72 transcript species in RAN translation.
  • To explore potential therapeutic targets by elucidating the mechanism of DPR production.

Main Methods:

  • Utilized long-read 5' RNA ligase-mediated rapid amplification of cDNA ends (5' Repeat-RLM-RACE) to identify C9orf72 transcripts.
  • Employed reporter assays to assess the translation efficiency of different intronic and exonic C9orf72 transcripts.
  • Manipulated lariat RNA stability by knocking down the Dbr1 enzyme in patient-derived iNeurons.

Main Results:

  • Identified cryptic, intron 1-initiating C9orf72 mRNAs (m7G-capped and polyadenylated) in mouse models and human neurons.
  • Demonstrated that these intron-initiating transcripts are more abundant and efficiently translated into DPRs compared to other potential templates like intron retention or lariat RNAs.
  • Found that enhancing lariat RNA stability did not affect DPR production, suggesting a minimal role for lariat RNAs in endogenous DPR synthesis.

Conclusions:

  • Cryptic, linear, intron-initiating C9orf72 mRNAs serve as the primary endogenous template for RAN translation and DPR production.
  • These findings provide critical insights into the molecular pathogenesis of C9orf72-associated ALS and FTD.
  • The identification of this novel RNA template opens new avenues for therapeutic strategies targeting C9orf72-mediated neurodegeneration.