Chromatin-dependent cryptic promoters encode alternative protein isoforms in yeast.
Cryptic transcription is widespread and generates a heterogeneous group of RNA molecules of unknown function. To improve our understanding of cryptic transcription, we investigated their transcription start site usage, chromatin organization and post-transcriptional consequences in Saccharomyces cerevisiae. We show that transcription start sites (TSSs) of chromatin-sensitive internal cryptic transcripts retain comparable features of canonical TSSs in terms of DNA sequence, directionality and chromatin accessibility. We degine the 5 and 3 boundaries of cryptic transcripts and show that, contrary to RNA degradation-sensitive ones, they often overlap with the end of the gene thereby using the canonical polyadenylation site and associate to polyribosomes. We show that chromatin-sensitive cryptic transcripts can be recognized by ribosomes and may produce truncated polypeptides from downstream, in-frame start codons. Finally, we congirm the presence of the predicted polypeptides by reanalyzing N-terminal proteomic datasets. Our work suggests that a fraction of chromatin-sensitive internal cryptic promoters are in fact alternative truncated mRNA isoforms. The expression of these chromatin-sensitive isoforms is conserved from yeast to human expanding the functional consequences of cryptic transcription and proteome complexity.