Cellular mRNAs access second ORFs using a novel amino acid sequence-dependent coupled translation termination–reinitiation mechanism

Abstract

Polycistronic transcripts are considered rare in the human genome. Initiation of translation of internal ORFs of eukaryotic genes has been shown to use either leaky scanning or highly structured IRES regions to access initiation codons. Studies on mammalian viruses identified a mechanism of coupled translation termination-reinitiation that allows translation of an additional ORF. Here, the ribosome terminating translation of ORF-1 translocates upstream to reinitiate translation of ORF-2. We have devised an algorithm to identify mRNAs in the human transcriptome in which the major ORF-1 overlaps a second ORF capable of encoding a product of at least 50 aa in length. This identified 4368 transcripts representing 2214 genes. We investigated 24 transcripts, 22 of which were shown to express a protein from ORF-2 highlighting that 3 ′ UTRs contain protein-coding potential more frequently than previously suspected. Five transcripts accessed ORF-2 using a process of coupled translation termination-reinitiation. Analysis of one transcript, encoding the CASQ2 protein, showed that the mechanism by which the coupling process of the cellular mRNAs was achieved was novel. This process was not directed by the mRNA sequence but required an aspartate-rich repeat region at the carboxyl terminus of the terminating ORF-1 protein.Introduction of wobble mutations for the aspartate codon had no effect, whereas replacing aspartate for glutamate repeats eliminated translational coupling. This is the first description of a coordinated expression of two proteins from cellular mRNAs using a coupled translation termination-reinitiation process and is the first example of such a process being determined at the amino acid level.

Global analysis of RNA cleavage by 5′-hydroxyl RNA sequencing

“…These signals were more prominent than any regions composed of any other single amino acid. Polyacidic stretches in the C-terminus of several human proteins are associated with ribosome frameshifting in these regions ( 55 ). However, it is not clear whether frameshifting is a consequence of the polyacidic nascent peptide, or whether it is a consequence of the propensity for out-of-frame polyglutamate codons to encode new Met initiation codons.…”

Two Alternative Ways of Start Site Selection in Human Norovirus Reinitiation of Translation

“…The prototype of this mechanism has been described for caliciviruses (23,24). Recently, a first case of coupled translation termination/reinitiation relying on a novel mechanism was found in cellular mRNAs (49).…”

Genome-Wide Association Study Identifies Phospholipase C zeta 1 (PLCz1) as a Stallion Fertility Locus in Hanoverian Warmblood Horses

“…Even if most of the eukaryotic mRNAs contain only one ORF, translation can be initiated at a downstream start codon due to leaky scanning [79] , [80] . Leaky scanning skips the major ORF and enables initiation of translation of internal second ORFs [81] .…”