Following the early demonstration by Steitz of ribosome footprints at the initiation codons of bacteriophage R17 RNA [6
], Wolin and Walter showed that eukaryotic ribosomes carrying out translation protected around 30 nucleotides of mRNA sequence from digestion by RNase [7
]. Exploiting this observation, they demonstrated clusters of ribosome protection at discrete sites in the preprolactin transcript. These clusters were interpreted as reflecting rate-limiting steps at translation initiation and termination, as well as ribosome pausing at the site of interaction of the nascent signal peptide with the signal recognition particle.
Ingolia et al
] have now extended analysis of these ribosome-protected fragments to the genome-wide scale through RNAseq technology. They implemented an imaginative intramolecular ligation strategy to generate directional, unbiased cDNA libraries for sequencing ribosome-protected RNA fragments. Despite significant contamination by ribosomal RNA, they were able to assign 7 × 106
RNAseq reads to more than 4,500 yeast genes. These ribosome 'footprints' were mapped with a high degree of precision and revealed a remarkable three-base periodicity corresponding to the codons within protein-coding sequences across the transcriptome. The abundance of ribosome-protected fragments from a given gene was used to predict the level of the encoded protein and was shown to be a significantly better predictor than mRNA level (multiple regression correlation coefficient R2
= 0.42 versus R2
This study also demonstrated how patterns of ribosome footprints could be used to provide insights into translational regulatory mechanisms. Figure illustrates potential sites of ribosome localization on a generic mRNA. From the Wolin and Walter study [7
], one would anticipate footprints at initiation codons and perhaps enhanced ribosome density at termination sites.
Figure 1 Positioning of ribosomes on a messenger RNA. The 5' cap is to the left and the poly(A) tail is to the right. The red symbols depict non-random accumulation of ribosomes at an uORF, the initiation codon, a site of ribosome pausing within the coding sequence (more ...)
Ribosomes would be expected to distribute randomly across coding sequences, with the exception of the codon periodicity noted above. Non-random occurrences of footprints within coding sequences are interpreted as sites of translational pausing, for example those associated with rare codons or co-translational activities. Within the untranslated terminal regions (UTRs) of mRNA, footprints might be expected in association with functional upstream open reading frames (uORFs). Indeed, as expected, Ingolia et al
] find that 98.8% of the footprints mapped to coding sequences, with the remainder predominantly associated with uORFs in the 5' UTRs.
Although uORFs are known to participate in translational control [8
], the extent of their translation across a transcriptome has never been evaluated. To attempt this, Ingolia et al
] annotated a total of 1,048 candidate uORFs with AUG starts in the yeast transcriptome and found that 153 of these showed evidence of ribosome association under the growth conditions examined. Among these ribosome-associated uORFs was the gene GCN4
. Ribosome footprints over the four uORFs in GCN4
behaved upon amino acid starvation as predicted by the generally accepted model [9
] for regulation of this gene - uORF 1 is constitutively translated and there is a reciprocal relationship between translation of uORFs 2-4 and the main coding sequence that is controlled by amino acid starvation.
Interestingly, regulated ribosome loading, apparently originating from two non-AUG starts, was observed upstream of the known uORFs in the GCN4
5' UTR. Although the existence of uORFs with non-AUG initiation codons has been the subject of speculation, the presence of these in GCN4
, as well as in more than 1,600 other candidates highlighted by Ingolia et al
], gives fascinating hints of previously unrecognized modes of translational control.