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Thousands of novel translated open reading frames in humans inferred by ribosome footprint profiling

View Article: PubMed Central - PubMed

ABSTRACT

Accurate annotation of protein coding regions is essential for understanding how genetic information is translated into function. We describe riboHMM, a new method that uses ribosome footprint data to accurately infer translated sequences. Applying riboHMM to human lymphoblastoid cell lines, we identified 7273 novel coding sequences, including 2442 translated upstream open reading frames. We observed an enrichment of footprints at inferred initiation sites after drug-induced arrest of translation initiation, validating many of the novel coding sequences. The novel proteins exhibit significant selective constraint in the inferred reading frames, suggesting that many are functional. Moreover, ~40% of bicistronic transcripts showed negative correlation in the translation levels of their two coding sequences, suggesting a potential regulatory role for these novel regions. Despite known limitations of mass spectrometry to detect protein expressed at low level, we estimated a 14% validation rate. Our work significantly expands the set of known coding regions in humans.

Doi:: http://dx.doi.org/10.7554/eLife.13328.001

No MeSH data available.


Related in: MedlinePlus

Validation of translated sequences identified in pseudogenes.Enrichment of harringtonine-arrested ribosome occupancy at the inferred translation initiation sites validates our inferred mCDS in pseudogenes.DOI:http://dx.doi.org/10.7554/eLife.13328.018
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fig4s1: Validation of translated sequences identified in pseudogenes.Enrichment of harringtonine-arrested ribosome occupancy at the inferred translation initiation sites validates our inferred mCDS in pseudogenes.DOI:http://dx.doi.org/10.7554/eLife.13328.018

Mentions: We next sought to provide independent experimental validation for the novel mCDS. A direct approach to validate translation initiation sites is to assay ribosome occupancy in cells treated with harringtonine (Ingolia et al., 2011). Harringtonine interacts with and arrests the initiation complex while leaving the elongation complex to continue translating and run off the transcript. Harringtonine-treated ribosome footprint profiling data therefore show a specific enrichment pattern at the translation initiation site; this pattern has previously been used to identify translation initiation sites in mouse embryonic stem cells (Ingolia et al., 2011). We measured harringtonine-treated ribosome footprints in two LCLs and aggregated the counts of footprints across all novel mCDS. We observed an enrichment of footprints at the inferred initiation site of the novel mCDS (binomial test, p-value = 9.5 × 10−79; Figure 4), similar to the enrichment of aggregate ribosome occupancy at the initiation sites of a matched number of mCDS that agreed exactly with the annotated CDS (see Figure 4—figure supplement 1 for mCDS in pseudogenes). We observed a significant enrichment at both AUG (p-value = 5.2 × 10−79) and non-AUG (p-value = 9.4 × 10−25) initiation sites. The reduced enrichment for the novel mCDS compared to annotated CDSs is likely due to the lower levels of translation of these novel mCDS and the high error rate in identifying the precise base at which translation is initiated. Accounting for these limitations, our observation of enrichment suggests that ribosomes do initiate the translation of many of the novel mCDS identified by riboHMM.10.7554/eLife.13328.017Figure 4.Validation of novel mCDS using harringtonine-treated ribosome profiling data.


Thousands of novel translated open reading frames in humans inferred by ribosome footprint profiling
Validation of translated sequences identified in pseudogenes.Enrichment of harringtonine-arrested ribosome occupancy at the inferred translation initiation sites validates our inferred mCDS in pseudogenes.DOI:http://dx.doi.org/10.7554/eLife.13328.018
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940163&req=5

fig4s1: Validation of translated sequences identified in pseudogenes.Enrichment of harringtonine-arrested ribosome occupancy at the inferred translation initiation sites validates our inferred mCDS in pseudogenes.DOI:http://dx.doi.org/10.7554/eLife.13328.018
Mentions: We next sought to provide independent experimental validation for the novel mCDS. A direct approach to validate translation initiation sites is to assay ribosome occupancy in cells treated with harringtonine (Ingolia et al., 2011). Harringtonine interacts with and arrests the initiation complex while leaving the elongation complex to continue translating and run off the transcript. Harringtonine-treated ribosome footprint profiling data therefore show a specific enrichment pattern at the translation initiation site; this pattern has previously been used to identify translation initiation sites in mouse embryonic stem cells (Ingolia et al., 2011). We measured harringtonine-treated ribosome footprints in two LCLs and aggregated the counts of footprints across all novel mCDS. We observed an enrichment of footprints at the inferred initiation site of the novel mCDS (binomial test, p-value = 9.5 × 10−79; Figure 4), similar to the enrichment of aggregate ribosome occupancy at the initiation sites of a matched number of mCDS that agreed exactly with the annotated CDS (see Figure 4—figure supplement 1 for mCDS in pseudogenes). We observed a significant enrichment at both AUG (p-value = 5.2 × 10−79) and non-AUG (p-value = 9.4 × 10−25) initiation sites. The reduced enrichment for the novel mCDS compared to annotated CDSs is likely due to the lower levels of translation of these novel mCDS and the high error rate in identifying the precise base at which translation is initiated. Accounting for these limitations, our observation of enrichment suggests that ribosomes do initiate the translation of many of the novel mCDS identified by riboHMM.10.7554/eLife.13328.017Figure 4.Validation of novel mCDS using harringtonine-treated ribosome profiling data.

View Article: PubMed Central - PubMed

ABSTRACT

Accurate annotation of protein coding regions is essential for understanding how genetic information is translated into function. We describe riboHMM, a new method that uses ribosome footprint data to accurately infer translated sequences. Applying riboHMM to human lymphoblastoid cell lines, we identified 7273 novel coding sequences, including 2442 translated upstream open reading frames. We observed an enrichment of footprints at inferred initiation sites after drug-induced arrest of translation initiation, validating many of the novel coding sequences. The novel proteins exhibit significant selective constraint in the inferred reading frames, suggesting that many are functional. Moreover, ~40% of bicistronic transcripts showed negative correlation in the translation levels of their two coding sequences, suggesting a potential regulatory role for these novel regions. Despite known limitations of mass spectrometry to detect protein expressed at low level, we estimated a 14% validation rate. Our work significantly expands the set of known coding regions in humans.

Doi:: http://dx.doi.org/10.7554/eLife.13328.001

No MeSH data available.


Related in: MedlinePlus