Evidence for RNA translatability, regulatory uORFs, and alternatively translated mRNAs. (A) (Top) The translation efficiency (TE) of lncRNAs and TUCPs distinguishes their translatability from that of 5′UTRs or CDSs of any size. (Bottom) The ribosome release score (RRS) across all possible ORFs separates lncRNAs and TUCPs from translated CDSs of any size. (B) Most erythroid lncRNAs and TUCPs show no evidence of translation. An empirical criterion for productive translation (supplemental Methods) classifies most lncRNAs and TUCPS, such as lincRNA-EPS and TCONS_00050143 (left panels), as noncoding. Others, such as Redrum and NR_015608 (right panels), are predicted to possess translated ORFs (depicted in red). (C) Translation of 5′UTR initiating ORFs. RFPs delineate the starts of high-confidence uORFs and exhibit 3-nt codon periodicity, evidencing their translation. (D) Validation of regulatory uORFs. Transient reporter assays (left, reporter design) evidence uORF-mediated translational control of the Bcl11A and Trak2 mRNAs (right). (E) Examples of uORFs in the Tal1 (left) and Bcl11a (right) mRNAs. (F) Examples of N-terminally extended proteins. Dashed lines mark annotated and upstream start codons. (G) Stop-codon-readthrough mRNA translation. RFPs continuing past known CDSs maintain 3-nt codon periodicity, evidencing readthrough translation. (H) Examples of C-terminally extended proteins, SAFB2 and RAPGEF1 (Rap guanine nucleotide exchange factor 1). Dashed lines mark annotated and next in-frame stop codons. CMV, cytomegalovirus; Fluc, firefly luciferase; Gabarap, GABA(A) receptor–associated protein; Mut, mutant allele; Rluc, Renilla luciferase; WT, wild-type allele.