NMD & Transcript Fate

Predicting Nonsense-Mediated Decay susceptibility and truncation severity.

Nonsense-mediated decay (NMD) degrades mRNA transcripts whose stop codon sits upstream of the last exon-exon junction. SPLISOFORMS predicts NMD susceptibility based on established biological rules and tracks whether surviving isoforms are significantly truncated compared to their canonical counterparts.

The 50-Nucleotide Rule

An isoform is classified as an NMD target if its stop codon lies more than 50 nt upstream of the final exon-exon junction (EJC). The two major escape exceptions are:

  • 5'-proximal escape: stop codon within ~150 nt of the start codon.
  • Single-exon transcripts: lack EJCs and naturally escape NMD.
NMD Target

Stop codon > 50 nt upstream of last EJC; not in 5'-proximal zone.

Result: mRNA degraded; protein not produced.

NMD Escape

Stop in last exon, within 50 nt of last EJC, or in 5'-proximal zone.

Result: transcript translated.

Status Values

NMD Target

50-nt rule applied: PTC > 50 nt upstream of the last EJC, not in the 5'-proximal escape zone.

NMD Escape

50-nt rule applied: stop in the last exon, within 50 nt of the last EJC, OR within ~150 nt of the CDS start (5'-proximal escape). Single-exon transcripts also land here (no EJC).

Non-Translatable

ORF type from SQANTI3 indicates no complete translatable sequence.

Predicted Translatable

Heuristic verdict used when no exact CDS coordinates are available. ORF looks complete, but we cannot positively rule out NMD without definitive CDS data.

Unknown

The 50-nt rule could not be applied due to missing exon coordinates or because the transcript is a smORF/uORF candidate.

Truncation Severity

Protein-length divergence vs. the canonical sequence is reported astruncation_severity. A short isoform may have a legitimate alternative TSS without triggering NMD, whereas a full-length isoform could still harbor a PTC.

Intact

≥ 90% of canonical length.

Minor

70 ≤ ratio < 90% (e.g. alternative TSS).

Severe

ratio < 70%. Suggests a PTC or major exon skip.

Non-translatable

No complete ORF available.

Microproteins & uORF Candidates

Because the 50-nt rule was not derived for short translated ORFs, they are systematically misclassified by the naïve rule. We flag these candidates separately and route them tonmd_status = unknown.

is_candidate_smorf

Isoform < 100 aa and canonical < 150 aa. May encode bona fide microproteins.

is_candidate_uorf

Isoform < 50 aa with a long canonical (≥ 150 aa). Heuristic flag for NMD-resistant upstream ORFs.

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