Mutations in HNRNPH1 prevent negative regulation via nonsense-mediated decay. We separately quantified canonical and alternative HNRNPH1 (A) and SRSF3 (B) transcripts by digital PCR in MCL cells (REC1, JVM2, and Z138) and HEK cells cultured with cycloheximide (an indirect inhibitor of NMD). This revealed an increasing proportion of the alternative transcript (skipped exon 4 in HNRNPH1) with increasing concentrations of cycloheximide. (C) Output from the minigene reporter is represented schematically. Differential splicing of the transcribed pre-mRNA results in inclusion or exclusion of HNRNPH1 exon 4. The out-of-frame transcript (right) results in the introduction of a premature termination codon (PTC) in exon 5 and translation of a truncated peptide. Translated peptides are represented in cartoon form. (D) HA tag abundance from the wild-type HNRNPH1 minigene is detected by western blot analysis. HA expression, representing productive splicing, is lost in the presence of HNRNPH1 overexpression. (E) The 3 independent G>T mutations introduced into the HNRNPH1 minigene are schematically represented in relation to the patient-identified mutations. (F) Expression of the HA tag from wild-type and mutant HNRNPH1 minigenes are detected by western blot analysis. HA expression, again representing productive splicing, is substantially higher when mutated minigenes are transfected into HEK cells as compared with the wild-type minigene.