Figure 6.

Dual coding in alternatively spliced PHPT1 exon. (A) Subcodon profile and mRNA-seq for PHPT1 mRNA variant NM_001135861 (left) and variant NM_014172 (right). (B) ORF organization for NM_001135861 (left) and NM_014172 (right). (C) Analysis of codon substitutions within the multiple alignments of orthologous genomic sequences for NM_001135861. (D) Exon organization of the two PHPT1 mRNA variants. For notations, see legend to Figure 4. Subcodon profiles for variant NM_001135861 (panel A, left), which is the longest isoform (see Methods), indicate that while the translated frame is the same as the CDS for most of the CDS region (low RPFs density for the second [red] position), the sequence is translated in the +1 frame relative to the CDS frame at its end and downstream (pink shaded area). In addition, there is an evident gap in translation in the subcodon profile and mRNA-seq just prior to the pink shaded area, which corresponds to the third exon in PHPT1 mRNA variant NM_001135861 (panel D). As a result, the fourth exon in the NM_001135861 mRNA is in an alternative frame relative to the CDS start codon. Codon substitution analysis of multiple sequence alignments (panel C) is consistent with the dual decoding of the 5′ end of the fourth exon. Synonymous and positive nonsynonymous substitutions are predominant in both the zero and +1 frames in the locations where RPFs are found.