Although ribosome-profiling and translation initiation sequencing (TI-seq) analyses have identified many noncanonical initiation codons, the precise detection of translation initiation sites (TISs) remains a challenge, mainly because of experimental artifacts of such analyses. Here, we describe a new method, TISCA (TIS detection by translation Complex Analysis), for the accurate identification of TISs. TISCA proved to be more reliable for TIS detection compared with existing tools, and it identified a substantial number of near-cognate codons in Kozak-like sequence contexts. Analysis of proteomics data revealed the presence of methionine at the NH2-terminus of most proteins derived from near-cognate initiation codons. Although eukaryotic initiation factor 2 (eIF2), eIF2A and eIF2D have previously been shown to contribute to translation initiation at near-cognate codons, we found that most noncanonical initiation events are most probably dependent on eIF2, consistent with the initial amino acid being methionine. Comprehensive identification of TISs by TISCA should facilitate characterization of the mechanism of noncanonical initiation.

An integrative understanding of nuclear events including transcription in normal and cancer cells requires comprehensive and quantitative measurement of protein dynamics that underlie such events. However, the low abundance of most nuclear proteins hampers their detailed functional characterization. We have now comprehensively quantified the abundance of nuclear proteins with the use of proteomics approaches in both normal and transformed human diploid fibroblasts. We found that subunits of the 26S proteasome complex were markedly down-regulated in the nuclear fraction of the transformed cells compared with that of the wild-type cells. The intranuclear proteasome abundance appeared to be inversely related to the rate of cell cycle progression, with restraint of the cell cycle being associated with an increase in the amount of proteasome subunits in the nucleus, suggesting that the nuclear proteasome content is dependent on the cell cycle. furthermore, chromatin enrichment for proteomics (chep) analysis revealed enrichment of the proteasome in the chromatin fraction of quiescent cells and its apparent dissociation from chromatin in transformed cells. our results thus suggest that translocation of the nuclear proteasome to chromatin may play an important role in control of the cell cycle and oncogenesis through regulation of chromatin-associated transcription factors. Most biological processes including development, cell differentiation and proliferation, and homeostasis in mammals are regulated at the level of gene expression. A full understanding of such processes will thus require comprehensive characterization of how the expression of each gene is regulated 1. The combination of genomics, proteomics, and other molecular technologies with bioinformatics has recently led to a rapid increase in our knowledge of gene regulatory networks that control gene expression 2-4. Proteomics is an indispensable technique for characterization of the dynamics of proteins. Given that the abundance of nuclear proteins that contribute to the control of gene expression is generally low, however, the application of proteomics to characterization of the dynamics of such proteins has been limited. Various methods to enrich nuclear proteins have been developed in an attempt to overcome this limitation. Although subcellular fractionation is a typical approach that has long been applied to separate the cytoplasm and nucleus with the use of hypotonic buffers and detergents, it is not suitable for isolation of proteins that bind to chromatin. Chromatin enrichment for proteomics (ChEP) enriches interphase chromatin and allows quantitative analysis of chromatin binding proteins. This approach is based on the cross-linking of intranuclear proteins to DNA in hypotonic buffer followed by isolation of the protein-DNA complex by centrifugation as a transparent gelatinous pellet 5. Another approach to nuclear protein enrichment based on the interaction of such proteins with synthetic DNA containing transcription factor response elements has been d...

Translation initiates when the eIF4F complex binds the 5 0 mRNA cap, followed by 5 0 untranslated region scanning for the start codon by scanning ribosomes. Here, we demonstrate that the ASC-1 complex (ASCC), which was previously shown to promote the dissociation of colliding 80S ribosomes, associates with scanning ribosomes to regulate translation initiation. Selective translation complex profiling (TCP-seq) analysis revealed that ASCC3, a helicase domaincontaining subunit of ASCC, localizes predominantly to the 5 0 untranslated region of mRNAs. Ribo-seq, TCP-seq, and luciferase reporter analyses showed that ASCC3 knockdown impairs 43S preinitiation complex loading and scanning dynamics, thereby reducing translation efficiency. Whereas eIF4A, an RNA helicase in the eIF4F complex, is important for global translation, ASCC was found to regulate the scanning process for a specific subset of transcripts. Our results have thus revealed that ASCC is required not only for dissociation of colliding 80S ribosomes but also for efficient translation initiation by scanning ribosomes at a subset of transcripts.