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Discovery of non-canonical translation initiation sites through mass spectrometric analysis of protein N-termini

ABSTRACT: Translation initiation generally occurs at AUG codons in eukaryotes although it has been shown that non-AUG or non-canonical translation initiation can also occur. However, the evidence for non-canonical translation initiation sites (TISs) is largely indirect and based on ribosome profiling studies. Here, using a strategy specifically designed to enrich N-termini of proteins, we demonstrate that many human proteins are translated at non-canonical TISs. The large majority of TISs that mapped to 5’ untranslated regions were non-canonical and led to N-terminal extension of annotated proteins or translation of upstream small open reading frames (uORF). There has been little controversy on whether the corresponding amino acid to the start codon is incorporated at TIS or methionine is still incorporated. Notably, methionine was incorporated at almost all non-canonical TISs identified in this study. Comparison of the TISs determined through mass spectrometry with ribosome profiling data revealed that about two-thirds of the novel annotations were indeed supported by ribosome profiling data. The sequence orthology analysis and relative translation frequency analysis of non-canonical TISs over canonical ones suggests that those non-canonical TISs are not leaky but they can have certain biological functions. Overall, this study provides evidence of protein translation initiation at non-canonical TISs and indicates that further studies are required for elucidation of functional implications of such non-canonical translation initiation.

INSTRUMENT(S): LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Endothelial Cell Of Umbilical Vein, Brain, Primary Cell, Permanent Cell Line Cell, Cell Culture, Colon

SUBMITTER: chanhyun na

LAB HEAD: Akhilesh Pandey

PROVIDER: PXD006633 | Pride | 2017-11-23

REPOSITORIES: Pride

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Project description:Purpose: Ribosome profiling and RNA-Seq were used to map the location and abundance of translating ribosomes on mouse heart and skeletal muscle transcripts. Methods: Tissue was rapidly harvested and snap-frozen to minimize bias to the pool of translating ribosomes. RNA was prepared from a single homogenate for each tissue so that starting RNA populations for both libraries were closely matched. Homogenates were not clarified before RNase digestion to avoid loss of ribosomes associated with large molecular weight complexes, and RNA-Seq libraries were prepared after rRNA subtraction to avoid positional loss of 5’ reads. Trimmed reads from 50 cycles of Illumina single-end sequencing were mapped onto a non-redundant set of 18,499 mouse protein-coding RefSeq transcripts from the nuclear genome. Results: Mapped sequence reads to myosin, actin and the giant protein titin together account for ~20% of the total mRNA-derived ribosome protected fragments (RPFs). We observed large-scale uniformity in the distribution of RPFs on the >30,000 codon titin open reading frame, from which we inferred an in vivo ribosome elongation error rate of ≤10-5. Ribosome footprints on Ttn mRNA also uncovered a novel 5’ UTR within a phylogenetically conserved intronic element that would produce ~2.35 mDa titin isoform that corresponds to the titin 'T2' band frequently described as a proteolytic artifact. Local translation efficiency across several >10 kb muscle mRNAs was also uniform, while their global translation efficiencies varied by ~20-fold suggesting initiation rate plays a major role in the translation efficiency of large mRNAs. Evidence for RPFs on 5’ UTRs was widespread with particular enrichment for ribosomes positioned at CUG codons. Comparison of global translation efficiency in cardiac and skeletal muscle revealed novel examples of tissue-specific translational control including synthesis of the myogenic factor Mef2c, and the titin-binding stress response protein Ankrd23. Conclusions: Our study represents the first detailed analysis of translation in an adult mammalian tissue generated by ribosome profiling technology. Current limitations to using ribosomal profiling in tissues include unknown perturbations to the dynamic state of translation despite rapidly harvested and snap-frozen samples. The uniform 5’ to 3’ coverage observed on individual large mRNAs and the ability to observe footprints on the extremely small phospholamban coding sequence, suggests that initiation and elongation were halted on similar time scales. More detailed examination of the positional information within CDS region requires further understanding of the bias introduced during the library preparation steps for both RPF-and RNA-Seq, as well as local biases induced as translation is arrested. Despite these qualifications, this initial view of active translation in muscle tissue highlights the potential for ribosome profiling to monitor the dynamic translation response to exercise, injury or disease pathology in animal models at a level of resolution not easily attainable with other quantitative approaches. Heart and skeletal muscle ribosome-protected fragment and RNA-Seq profiles of 10-week old C57BL/6J male mice were generated by deep sequencing using the Illumina HiSeq 2000.

Project description:Purpose: Ribosome profiling and RNA-Seq were used to map the location and abundance of translating ribosomes on human skeletal muscle transcripts from a patient with Becker muscular dystrophy. Methods: Tissue homogenates were prepared from frozen sections of a muscle biopsy obtained from a patient with an NM_004006:c.40_41delGA dystrophin mutation and a normal control. Ribosome-protected fragments and total RNA were prepared from a single homogenate, so starting RNA populations for both libraries were closely matched. Homogenates were not clarified before RNase digestion to avoid loss of ribosomes associated with large molecular weight complexes and RNA-Seq libraries were prepared after rRNA subtraction to avoid positional loss of 5’ reads. RPF-Seq libraries were built using the TruSeq Small RNA Sample Preparation Kit (Illumina) and RNA-Seq libraries were built using the TruSeq RNA Sample Preparation v2 Kit (Illumina). RPF-Seq and RNA-Seq libraries were subjected to 50 cycles of single-end sequencing on an Illumina HiSeq 2000 instrument. Trimmed and filtered RPF- and RNA-Seq reads were mapped to RefSeq fasta sequences downloaded from the UCSC genome browser (hg19 assembly). Results: Most mutations that truncate the reading frame of the DMD gene result in loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity can result from alternate translation initiation beginning in DMD exon 6 that results in the expression of a highly functional N-truncated dystrophin. This novel isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid-inducible. IRES activity is confirmed in patient muscle by both peptide sequencing and ribosomal profiling. Conclusions: Our results provide a molecular explanation for the rescue of 5’ truncating mutations via a heretofore undescribed mechanism of post-transcriptional regulation of dystrophin expression. The presence of a glucocorticoid-inducible IRES within a highly conserved region of the DMD sequence strongly suggests a programmed role for alternate translation initiation, and ongoing efforts to understand the relevant cell lineage-specific and/or conditional activation signals will shed light on underlying mechanisms of IRES control and elucidate potentially novel functions of dystrophin. Skeletal muscle ribosome-protected fragment and RNA-Seq profiles from a patient with an NM_004006:c.40_41delGA dystrophin mutation and a normal control were generated by deep sequencing using the Illumina HiSeq 2000.

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Discovery of non-canonical translation initiation sites through mass spectrometric analysis of protein N-termini

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