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Deep sequencing of ribosome footprints (ribosome profiling) maps and
quantifies mRNA translation. Because ribosomes decode mRNA every 3 nt, the
periodic property of ribosome footprints could be used to identify novel
translated ORFs. However, due to the limited resolution of existing
methods, the 3-nt periodicity is observed mostly in a global analysis, but
not in individual transcripts. Here, we report a protocol applied to
Arabidopsis that maps over 90% of the footprints to the main reading frame
and thus offers super-resolution profiles for individual transcripts to
precisely define translated regions. The resulting data not only support
many annotated and predicted noncanonical translation events but also
uncover small ORFs in annotated noncoding RNAs and pseudogenes. A
substantial number of these unannotated ORFs are evolutionarily conserved,
and some produce stable proteins. Thus, our study provides a valuable
resource for plant genomics and an efficient optimization strategy for
ribosome profiling in other organisms.
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