Conformational rearrangements upon start codon recognition in human 48S translation initiation complex

Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1, eIF1A, eIF2–GTP–Met-tRNAiMet, and eIF3. The ‘open’ 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step. The ‘closed’ form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast.

TIS transformer: Re-annotation of the human proteome using deep learning

The precise detection of translation initiation sites is essential for proteome delineation. In turn, the accurate mapping of the proteome is fundamental in advancing our understanding of biological systems and cellular mechanisms. We propose TIS Transformer, a deep learning model for the determination of translation start sites, based on information embedded in processed transcript nucleotide sequences. Through the application of deep learning techniques first designed for natural language processing tasks, we have developed an approach that achieves state-of-the-art performances on the prediction of translation initiation sites. TIS Transformer utilizes the FAVOR+ algorithm for attention calculation, enabling processing of full transcript sequences by the model. Analysis of input importance revealed TIS Transformer's ability to detect key features of translation, such as translation stop sites and reading frames. Furthermore, we demonstrate TIS Transformer's ability to detect multiple peptides on a transcript, and peptides encoded by short Open Reading Frames (sORFs), either alongside a canonical coding sequence or in long non-coding RNAs. Using a cross-validation scheme, we apply TIS Transformer to re-annotate the full human transcriptome.

Elucidating the Importance and Regulation of Key Enhancers for Human MEIS1 Expression

Myeloid ecotropic virus insertion site 1 (MEIS1) is essential for normal hematopoiesis and is deregulated in a large subset of acute myeloid leukemia (AML) by yet unknown mechanisms. We previously identified 3 candidate enhancer regions: enhancer region 1 (E1) at -2 kb upstream; enhancer region 2 (E2) at +10.6 kb downstream inside intron 6; and enhancer region 3 (E3) +140 kb downstream of the translation start site. In the current study, we utilized CRISPR-Cas9 genome editing to further characterize these enhancers in a human AML cell line and identify the key transcription factors (TFs) associated with their function. To efficiently track MEIS1 expression levels, a GFP reporter, a P2A self-cleaving peptide tag and a hemagglutinin tag at its translation start site was introduced in a MEIS1 high expressing human AML cell line, U937. Then we introduced random mutations (Indels) along the MEIS1 locus utilizing a CRISPR-Cas9 mediated genome editing vector system in mono-allelic MEIS1-GFP-tagged U937 cells with special focus on the previously identified enhancer regions to find the key sequences important to the function of the MEIS1 enhancer regions. Two targeted regions yielding the highest proportion of GFP - cells corresponded to the E2 enhancer region within intron 6 and were referred to as E2.1 and E2.2. Using chromosome conformation capture (3C) assay, we detected a significantly decreased interaction (p=0.0022) between the promoter and the intron 6 region surrounding the E2 region in E2.2 targeted cells compared to the parental cells. Moreover, our data indicated that the DNA sequence within E2.2 is highly critical to this region's enhancer function which is further influenced by the larger genomic region surrounding the E2.1 gRNA targeted site. To identify TFs binding to the E2 region, we further scrutinized the E2.2 indel region for loss of TF binding sites. We performed TF prediction analysis and performed a protein pull down-mass spectrometry experiment to identify TF candidates. The overlap yielded a list of 7 TFs, each of which we targeted via CRISPR/Cas9. Reduction in GFP levels was only observed for FLI1 locus targeting but not for the other 6 TFs. Concordant reduction in MEIS1 and FLI1 levels were confirmed by immunoblotting. Additionally, chromatin immunoprecipitation (ChIP) followed by quantitative PCR revealed significant FLI1 enrichment at the promoter and at 3 sites surrounding the E2.2 region (p=0.0004) compared to 4 control regions scattered along the MEIS1 locus. Given a previous study indicating MEIS1 upregulation of FLI1 in normal hematopoiesis, we hypothesised that a positive feedback loop may exist between FLI1 and MEIS1 in AML. Since MEIS1 levels are frequently elevated in normal karyotype AML (CN-AML), we used the murine Hoxa9/Meis1 AML model as a surrogate for CN-AML and performed Meis1 ChIP-seq analysis. We detected direct Meis1 binding to the intronic region of the mouse Fli1 gene as well as other ETS factor loci, in Hoxa9/Meis1 cells. To better understand the clinical relevance of FLI1 in AML, we analyzed the Beat AML dataset. High FLI1 transcript levels correlated with adverse overall survival in CN-AML (p=0.044). Additionally, we observed a trend towards worse outcome with high FLI1 in the NPM1-mutated CN-AML subtype (p=0.069). We also observed a similar correlation in CN-AML for another ETS factor, ELF1, which we had previously shown to bind and upregulate MEIS1 expression in AML, suggesting a broader unrecognized role for ETS factors in AML. In summary, we have developed a rapid flow cytometry-based readout for the fine dissection and characterization of the cis-regulatory elements and associated TFs critical for MEIS1 transcription via CRISPR-Cas9 genetic manipulation. Our study revealed FLI1 as the candidate key regulator of MEIS1 expression and a positive correlation between FLI1 mRNA levels and worse overall survival in MEIS1-high AML subgroups. Disclosures No relevant conflicts of interest to declare.

Monitoring RNA dynamics in native transcriptional complexes

Cotranscriptional RNA folding is crucial for the timely control of biological processes, but because of its transient nature, its study has remained challenging. While single-molecule Förster resonance energy transfer (smFRET) is unique to investigate transient RNA structures, its application to cotranscriptional studies has been limited to nonnative systems lacking RNA polymerase (RNAP)–dependent features, which are crucial for gene regulation. Here, we present an approach that enables site-specific labeling and smFRET studies of kilobase-length transcripts within native bacterial complexes. By monitoring Escherichia coli nascent riboswitches, we reveal an inverse relationship between elongation speed and metabolite-sensing efficiency and show that pause sites upstream of the translation start codon delimit a sequence hotspot for metabolite sensing during transcription. Furthermore, we demonstrate a crucial role of the bacterial RNAP actively delaying the formation, within the hotspot sequence, of competing structures precluding metabolite binding. Our approach allows the investigation of cotranscriptional regulatory mechanisms in bacterial and eukaryotic elongation complexes.

The Contribution of the Predicted Sorting Platform Component HrcQ to Type III Secretion in Xanthomonas campestris pv. vesicatoria Depends on an Internal Translation Start Site

Pathogenicity of the Gram-negative bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system which translocates effector proteins into plant cells. T3S systems are conserved in plant- and animal-pathogenic bacteria and consist of at least nine structural core components, which are designated Sct (secretion and cellular translocation) in animal-pathogenic bacteria. Sct proteins are involved in the assembly of the membrane-spanning secretion apparatus which is associated with an extracellular needle structure and a cytoplasmic sorting platform. Components of the sorting platform include the ATPase SctN, its regulator SctL, and pod-like structures at the periphery of the sorting platform consisting of SctQ proteins. Members of the SctQ family form a complex with the C-terminal protein domain, SctQC, which is translated as separate protein and likely acts either as a structural component of the sorting platform or as a chaperone for SctQ. The sorting platform has been intensively studied in animal-pathogenic bacteria but has not yet been visualized in plant pathogens. We previously showed that the SctQ homolog HrcQ from X. campestris pv. vesicatoria assembles into complexes which associate with the T3S system and interact with components of the ATPase complex. Here, we report the presence of an internal alternative translation start site in hrcQ leading to the separate synthesis of the C-terminal protein region (HrcQC). The analysis of genomic hrcQ mutants showed that HrcQC is essential for pathogenicity and T3S. Increased expression levels of hrcQ or the T3S genes, however, compensated the lack of HrcQC. Interaction studies and protein analyses suggest that HrcQC forms a complex with HrcQ and promotes HrcQ stability. Furthermore, HrcQC colocalizes with HrcQ as was shown by fluorescence microscopy, suggesting that it is part of the predicted cytoplasmic sorting platform. In agreement with this finding, HrcQC interacts with the inner membrane ring protein HrcD and the SctK-like linker protein HrpB4 which contributes to the docking of the HrcQ complex to the membrane-spanning T3S apparatus. Taken together, our data suggest that HrcQC acts as a chaperone for HrcQ and as a structural component of the predicted sorting platform.

utr.annotation: a tool for annotating genomic variants that could influence post-transcriptional regulation

Summary Whole genome sequencing of patient populations is identifying thousands of new variants in UnTranslated Regions(UTRs). While the consequences of UTR mutations are not as easily predicted from primary sequence as coding mutations are, there are some known features of UTRs that modulate their function. utr.annotation is an R package that can be used to annotate potential deleterious variants in the UTR regions for both human and mouse species. Given a CSV or VCF format variant file, utr.annotation provides information of each variant on whether and how it alters known translational regulators including: upstream Open Reading Frames (uORFs), upstream Kozak sequences, polyA signals, Kozak sequences at the annotated translation start site, start codons, and stop codons, conservation scores in the variant position, and whether and how it changes ribosome loading based on a model derived from empirical data. Availability utr.annotation is freely available on Bitbucket (https://bitbucket.org/jdlabteam/utr.annotation/src/master/) and CRAN (https://cran.r-project.org/web/packages/utr.annotation/index.html) Supplementary information Supplementary data are available at https://wustl.box.com/s/yye99bryfin89nav45gv91l5k35fxo7z.

Mutations of the Genomes Uncoupled 4 Gene Cause ROS Accumulation and Repress Expression of Peroxidase Genes in Rice

The Genomes Uncoupled 4 (GUN4) is one of the retrograde signaling genes in Arabidopsis and its orthologs have been identified in oxygenic phototrophic organisms from cyanobacterium to higher plants. GUN4 is involved in tetrapyrrole biosynthesis and its mutation often causes chlorophyll-deficient phenotypes with increased levels of reactive oxygen species (ROS), hence it has been speculated that GUN4 may also play a role in photoprotection. However, the biological mechanism leading to the increased ROS accumulation in gun4 mutants remains largely unknown. In our previous studies, we generated an epi-mutant allele of OsGUN4 (gun4epi), which downregulated its expression to ∼0.5% that of its wild-type (WT), and a complete knockout allele gun4-1 due to abolishment of its translation start site. In the present study, three types of F2 plant derived from a gun4-1/gun4epi cross, i.e., gun4-1/gun4-1, gun4-1/gun4epi and gun4epi/gun4epi were developed and used for further investigation by growing them under photoperiodic condition (16 h/8 h light/dark) with low light (LL, 100 μmol photons m–2 s–1) or high light (HL, 1000 μmol photons m–2 s–1). The expression of OsGUN4 was light responsive and had two peaks in the daytime. gun4-1/gun4-1-F2 seeds showed defective germination and died within 7 days. Significantly higher levels of ROS accumulated in all types of OsGUN4 mutants than in WT plants under both the LL and HL conditions. A comparative RNA-seq analysis of WT variety LTB and its gun4epi mutant HYB led to the identification of eight peroxidase (PRX)-encoding genes that were significantly downregulated in HYB. The transcription of these eight PRX genes was restored in transgenic HYB protoplasts overexpressing OsGUN4, while their expression was repressed in LTB protoplasts transformed with an OsGUN4 silencing vector. We conclude that OsGUN4 is indispensable for rice, its expression is light- and oxidative-stress responsive, and it plays a role in ROS accumulation via its involvement in the transcriptional regulation of PRX genes.

Transcriptional control of chicken KLF7 promoter in preadipocytes

Krüppel-like factor 7 (KLF7) has been reported to inhibit adipogenesis and regulate the development of the nervous system. However, transcription regulation of KLF7 remains poorly understood. In the current study, a 2196-bp-long 5′-flanking sequence of chicken KLF7 (−2286 bp to −91 bp, upstream of the translation start site) was studied for promoter activity, and there was a remarkable promoter activity in this sequence (P<0.05). The 5′-truncated mutation analysis showed that a minimal promoter was on the sequence from −241 bp to −91 bp. In addition, GATA2 overexpression facilitated the promoter activity of pGL3-KLF7(−2286/−91), pGL3-KLF7(−1215/−91), pGL3-KLF7(−521/−91), and pGL3-KLF7(−241/−91), and GATA3 overexpression inhibited the promoter activity of pGL3-KLF7(−1845/−91), pGL3-KLF7(−1215/−91), pGL3-KLF7(−521/−91), and pGL3-KLF7(−241/−91) in chicken preadipocytes (P<0.05). Knockdown of GATA2 expression inhibited the promoter activity of pGL3-KLF7(−1215/−91) and pGL3-KLF7(−241/−91), and knockdown of GATA3 expression facilitated the promoter activity of pGL3-KLF7(−521/−91) and pGL3-KLF7(−241/−91) (P<0.05). Additionally, overexpression and knockdown analyses showed that GATA3 inhibited KLF7 mRNA expression (P<0.05), and both overexpression and knockdown of GATA2 resulted in the downregulation of KLF7 mRNA expression in chicken preadipocytes (P<0.05). Western blot analysis in chicken preadipocytes showed that GATA2 facilitated KLF7 expression and GATA3 inhibited KLF7 expression. Mutation analysis showed that the motif of ‘GGATCTATCA’ (−107 bp/−98 bp) might be a cis-regulation element, which is involved in the KLF7 expression regulation by GATA3 in chicken preadipocytes. These results provided some details of KLF7 transcription regulation in chicken adipose tissue.