Alternative Open Reading Frame Research Articles

Proteins à la carte: riboproteogenomic exploration of bacterial N-terminal proteoform expression.

With the emerging theme of genes within genes comprising the existence of alternative open reading frames (ORFs) generated by translation initiation at in-frame start codons, mechanisms that control the relative utilization of annotated and alternative TIS need to be unraveled and our molecular understanding of resulting proteoforms broadened. Utilizing complementary ribosome profiling strategies to map ORF boundaries, we uncovered dual-encoding ORFs generated by in-frame TIS usage in Salmonella. Besides demonstrating that alternative TIS usage may generate proteoforms with different characteristics, such as differential localization and specialized function, quantitative aspects of conditional retapamulin-assisted ribosome profiling (Ribo-RET) translation initiation maps offer unprecedented insights into the relative utilization of annotated and alternative TIS, enabling the exploration of gene regulatory mechanisms that control TIS usage and, consequently, the translation of N-terminal proteoform pairs.

Evolving membrane-associated accessory protein variants for improved adeno-associated virus production

Manufacturing sufficient adeno-associated virus (AAV) to meet current and projected clinical needs is a significant hurdle to the growing gene therapy industry. The recently discovered membrane-associated accessory protein (MAAP) is encoded by an alternative open reading frame in the AAV cap gene that is found in all presently reported natural serotypes. Recent evidence has emerged supporting a functional role of MAAP in AAV egress, although the underlying mechanisms of MAAP function remain unknown. Here, we show that inactivation of MAAP from AAV2 by a single point mutation that is silent in the VP1 open reading frame (ORF) (AAV2-ΔMAAP) decreased exosome-associated and secreted vector genome production. We hypothesized that novel MAAP variants could be evolved to increase AAV production and thus subjected a library encoding over 1×106 MAAP protein variants to five rounds of packaging selection into the AAV2-ΔMAAP capsid. Between each successive packaging round, we observed a progressive increase in both overall titer and ratio of secreted vector genomes conferred by the bulk-selected MAAP library population. Next-generation sequencing uncovered enriched mutational features, and a resulting selected MAAP variant containing missense mutations and a frameshifted C-terminal domain increased overall GFP transgene packaging in AAV2, AAV6, and AAV9 capsids.

ORFeus: a computational method to detect programmed ribosomal frameshifts and other non-canonical translation events

BackgroundIn canonical protein translation, ribosomes initiate translation at a specific start codon, maintain a single reading frame throughout elongation, and terminate at the first in-frame stop codon. However, ribosomal behavior can deviate at each of these steps, sometimes in a programmed manner. Certain mRNAs contain sequence and structural elements that cause ribosomes to begin translation at alternative start codons, shift reading frame, read through stop codons, or reinitiate on the same mRNA. These processes represent important translational control mechanisms that can allow an mRNA to encode multiple functional protein products or regulate protein expression. The prevalence of these events remains uncertain, due to the difficulty of systematic detection.ResultsWe have developed a computational model to infer non-canonical translation events from ribosome profiling data.ConclusionORFeus identifies known examples of alternative open reading frames and recoding events across different organisms and enables transcriptome-wide searches for novel events.

Membrane-bound Merkel cell polyomavirus middle T protein constitutively activates PLCγ1 signaling through Src-family kinases.

Merkel cell polyomavirus (MCV or MCPyV) is an alphapolyomavirus causing human Merkel cell carcinoma and encodes four tumor (T) antigen proteins: large T (LT), small tumor (sT), 57 kT, and middle T (MT)/alternate LT open reading frame proteins. We show that MCV MT is generated as multiple isoforms through internal methionine translational initiation that insert into membrane lipid rafts. The membrane-localized MCV MT oligomerizes and promiscuously binds to lipid raft-associated Src family kinases (SFKs). MCV MT-SFK interaction is mediated by a Src homology (SH) 3 recognition motif as determined by surface plasmon resonance, coimmunoprecipitation, and bimolecular fluorescence complementation assays. SFK recruitment by MT leads to tyrosine phosphorylation at a SH2 recognition motif (pMTY114), allowing interaction with phospholipase C gamma 1 (PLCγ1). The secondary recruitment of PLCγ1 to the SFK-MT membrane complex promotes PLCγ1 tyrosine phosphorylation on Y783 and activates the NF-κB inflammatory signaling pathway. Mutations at either the MCV MT SH2 or SH3 recognition sites abrogate PLCγ1-dependent activation of NF-κB signaling and increase viral replication after MCV genome transfection into 293 cells. These findings reveal a conserved viral targeting of the SFK-PLCγ1 pathway by both MCV and murine polyomavirus (MuPyV) MT proteins. The molecular steps in how SFK-PLCγ1 activation is achieved, however, differ between these two viruses.

A Genomic Approach for High-Grade B Cell Lymphoma with MYC and BCL2 and / or BCL6 Rearrangements: Two Clusters Stood out on Next Generation Sequencing Results

Introduction High-grade B cell lymphomas with double or triple hit rearrangement (HGBL DH/TH), which appeared on the 4th revised edition WHO classification of lymphoid neoplasms, are aggressive mature B-cell lymphomas with high-grade cytology (Burkitt-like, blastoid or DLBCL-like) that have a MYC (8q24) rearrangement in combination with a BCL2 (18q21) and / or a BCL6 (3q27) rearrangement. There are currently no treatment guidelines for this subtype, and the usual immunochemotherapy leads to few complete remissions. While the NGS technology has been used in diffuse large B cell lymphoma, there has been no specific study on additional genetic events which would predict the clinical outcome in HGBL DH/TH. Here, we report our preliminary results using a targeted DNA capture panel approach (NGS) to identify subgroups with different clinical outcomes among HGBL DH/TH. Methods We conducted a retrospective study on 44 patients diagnosed with DH or TH HGBL and treated at Centre Hospitalier Lyon Sud or Centre Léon Bérard starting in 2013. We collected the medical information regarding the treatment, time to response to treatment or relapse, progression free survival, and overall survival. They all benefited from a DNA-targeted NGS analysis covering 54 genes. Results The cohort included 20 females and 24 males, with a median age of 65 (31-87). Among them, 22 patients were MYC/ BCL2 DH (50%), 12 were MYC/ BCL6 DH (27,3%) and 10 were TH (22,7%). Eleven patients had B symptoms (25%). The Ann Arbor stage was high (III-IV) in 97.5%. The WHO Performance Status was low (0-1) for most of the patients (70%). The majority of them presented with an IPI above to 2 (82,5%) and an elevated LDH level (80%). The Ki67 index was above 70% (33/42) in 78,5% of the cases and above 90% (19/42) in nearly half of them. The major finding of the NGS analysis was the identification of 2 groups based on the TP53/ CDKN2A_p14ARF mutational status. The CDKN2A locus encodes for 2 alternative open reading frames leading to 2 unrelated proteins p14ARF and p16INK4A. P14ARF is involved in the regulation of the TP53 pathway whereas p16INK4A is not. We therefor defined 2 groups : the first group included patients with either TP53 or CDKN2A_p14ARF-only alterations (group A, n=10) while the second group had no alteration in these two coding sequences (group B, n=34) although 12 patients had a CDKN2A_p16INK4A loss or mutation affecting both CDKN2A transcripts. Most of TP53 mutations were missense mutations mostly in TP53 DNA-binding domain and 2 cases appeared to be single hit mutations with an allele frequency below 50%. The patient with a p14ARF only mutation had a start codon mutation p.(M1*) with an allele frequency of 96% without CDKN2A copy number loss. The median overall survival (OS) (Figure 1) was 6,2 months for group A, and 55,5 months for group B (p = 0,0076). The median follow-up time was 14 months (7,5 months in the group A, 18 in group B). Only 2 patients in group A survived and are still in complete remission, one was DH BCL6 and the other DH BCL2 Conclusion Our preliminary results indicate a strong negative prognostic impact of either CDKN2A_p14ARF or TP53 gene alterations on the OS of HGBL DH/TH patients. The biological basis for the difference between groups would be in favour of the alteration of the p14ARF-TP53 tumor suppressive pathway as major contributor of chemoresistance. A more detailed analysis is on-going to further identify mutation profiles of double hit and triple hit lymphomas and compare our finding with existing data. Our results confirm the more favourable outcome of DH BCL6 patients (data not shown), which is consistent with the 5 th revised OMS classification that removed that specific entity. Our results also indicate that routine NGS analysis of HGBL DH/TH patients offers new perspectives for patient stratification. Regarding their survival, patients with TP53 pathway alterations should probably not be treated with the standard RCHOP chemo-immunotherapy. Instead, a more intensive immunochemotherapy regimen or novel strategies such as first-line bispecific antibodies or CARTCells might be considered.

OpenProt 2.0 builds a path to the functional characterization of alternative proteins.

The OpenProt proteogenomic resource (https://www.openprot.org/) provides users with a complete and freely accessible set of non-canonical or alternative open reading frames (AltORFs) within the transcriptome of various species, as well as functional annotations of the corresponding protein sequences not found in standard databases. Enhancements in this update are largely the result of user feedback and include the prediction of structure, subcellular localization, and intrinsic disorder, using cutting-edge algorithms based on machine learning techniques. The mass spectrometry pipeline now integrates a machine learning-based peptide rescoring method to improve peptide identification. We continue to help users explore this cryptic proteome by providing OpenCustomDB, a tool that enables users to build their own customized protein databases, and OpenVar, a genomic annotator including genetic variants within AltORFs and protein sequences. A new interface improves the visualization of all functional annotations, including a spectral viewer and the prediction of multicoding genes. All data on OpenProt are freely available and downloadable. Overall, OpenProt continues to establish itself as an important resource for the exploration and study of new proteins.

Azyx-1 is a new gene that overlaps with zyxin and affects its translation in C. elegans, impacting muscular integrity and locomotion.

Overlapping genes are widely prevalent; however, their expression and consequences are poorly understood. Here, we describe and functionally characterize a novel zyx-1 overlapping gene, azyx-1, with distinct regulatory functions in Caenorhabditis elegans. We observed conservation of alternative open reading frames (ORFs) overlapping the 5' region of zyxin family members in several animal species, and find shared sites of azyx-1 and zyxin proteoform expression in C. elegans. In line with a standard ribosome scanning model, our results support cis regulation of zyx-1 long isoform(s) by upstream initiating azyx-1a. Moreover, we report on a rare observation of trans regulation of zyx-1 by azyx-1, with evidence of increased ZYX-1 upon azyx-1 overexpression. Our results suggest a dual role for azyx-1 in influencing zyx-1 proteoform heterogeneity and highlight its impact on C. elegans muscular integrity and locomotion.

Heimdall, an alternative protein issued from a ncRNA related to kappa light chain variable region of immunoglobulins from astrocytes: a new player in neural proteome

The dogma “One gene, one protein” is clearly obsolete since cells use alternative splicing and generate multiple transcripts which are translated into protein isoforms, but also use alternative translation initiation sites (TISs) and termination sites on a given transcript. Alternative open reading frames for individual transcripts give proteins originate from the 5′- and 3′-UTR mRNA regions, frameshifts of mRNA ORFs or from non-coding RNAs. Longtime considered as non-coding, recent in-silico translation prediction methods enriched the protein databases allowing the identification of new target structures that have not been identified previously. To gain insight into the role of these newly identified alternative proteins in the regulation of cellular functions, it is crucial to assess their dynamic modulation within a framework of altered physiological modifications such as experimental spinal cord injury (SCI). Here, we carried out a longitudinal proteomic study on rat SCI from 12 h to 10 days. Based on the alternative protein predictions, it was possible to identify a plethora of newly predicted protein hits. Among these proteins, some presented a special interest due to high homology with variable chain regions of immunoglobulins. We focus our interest on the one related to Kappa variable light chains which is similarly highly produced by B cells in the Bence jones disease, but here expressed in astrocytes. This protein, name Heimdall is an Intrinsically disordered protein which is secreted under inflammatory conditions. Immunoprecipitation experiments showed that the Heimdall interactome contained proteins related to astrocyte fate keepers such as “NOTCH1, EPHA3, IPO13” as well as membrane receptor protein including “CHRNA9; TGFBR, EPHB6, and TRAM”. However, when Heimdall protein was neutralized utilizing a specific antibody or its gene knocked out by CRISPR-Cas9, sprouting elongations were observed in the corresponding astrocytes. Interestingly, depolarization assays and intracellular calcium measurements in Heimdall KO, established a depolarization effect on astrocyte membranes KO cells were more likely that the one found in neuroprogenitors. Proteomic analyses performed under injury conditions or under lipopolysaccharides (LPS) stimulation, revealed the expression of neuronal factors, stem cell proteins, proliferation, and neurogenesis of astrocyte convertor factors such as EPHA4, NOTCH2, SLIT3, SEMA3F, suggesting a role of Heimdall could regulate astrocytic fate. Taken together, Heimdall could be a novel member of the gatekeeping astrocyte-to-neuroprogenitor conversion factors.

SARS-CoV-2 ORF3c impairs mitochondrial respiratory metabolism, oxidative stress, and autophagic flux

Coronaviruses encode a variable number of accessory proteins that are involved in host-virus interaction, suppression of immune responses, or immune evasion. SARS-CoV-2 encodes at least twelve accessory proteins, whose roles during infection have been studied. Nevertheless, the role of the ORF3c accessory protein,an alternative open reading frame of ORF3a, has remained elusive. Herein, we show that the ORF3c protein has a mitochondrial localization and alters mitochondrial metabolism, inducing a shift from glucose to fatty acids oxidation and enhanced oxidative phosphorylation. These effects result in increased ROS production and block of the autophagic flux. In particular, ORF3c affects lysosomal acidification, blocking the normal autophagic degradation process and leading to autolysosome accumulation. We also observed different effect on autophagy for SARS-CoV-2 and batCoV RaTG13 ORF3c proteins; the 36R and 40K sites are necessary and sufficient to determine these effects.

The expression level of ARF and p53 in AML patients, and their relation to patients' outcome

BackgroundAcute myeloid leukemia (AML) is a cancer of hematopoietic progenitors characterized by gene mutations. The most popular deregulations are mutation and altered expression in the p53 gene, which is considered the guardian of the genome. Its activity is controlled by regulatory genes, e.g., alternate open reading frame (ARF), whose defects could affect p53 activity.AimTo study the effect of altered expression of p53 and ARF genes in de novo AML patients and correlate the results to the patients’ characteristics and outcomes.MethodsExpression levels of p53 and ARF were assessed in 96 AML adult patients compared to 20 healthy controls using quantitative reverse-transcription PCR (RT-qPCR).ResultsThere was significant up-regulation of p53 [77.6 (3.8–9528.3)] compared to controls [1.031 (0.210–9.051)], p < 0.001]. The expression level of ARF was significantly upregulated [6.2 (0.5–964.0)] compared to controls [0.854 (0.357–2.519), p < 0.001]. All of the low ARF expressers had low p53 overexpression, 61.1% of patients with high ARF expression had high p53 over-expression, and 38.9% with high ARF expression had low p53 over-expression (p < 0.001). ARF expression shows a trend of association with FLT3 mutation, as 89.3% with FLT3 mutation have high ARF expression (p = 0.080). Low p53 over-expression was seen in 77% of APL patients, while high p53 expression was associated with non-APL (p = 0.040). The median DFS of mutant NPM1 patients was higher than wild NPM1 (46.15 vs. 5.89 days, p = 0.045). Patients aged ≤ 50 years had better OS and DFS than those > 50 (p = 0.05, p = 0.035, respectively).There were no significant statistical associations between DFS and p53, ARF, and FLT3 mutations.ConclusionThe p53 and ARF genes are overexpressed in de novo AML patients and they are interrelated. low p53 overexpression is associated with APL phenotype and t(15;17) and patients with t(15;17) had slightly better survival than patients with negative t(15;17) (p = 0.061). AML patients with mutated NPM1 had better DFS than wild NPM1 (p = 0.045). p53 pathway regulation can occur by many alternative ways rather than gene mutation.

OpenCustomDB: Integration of Unannotated Open Reading Frames and Genetic Variants to Generate More Comprehensive Customized Protein Databases.

Proteomic diversity in biological samples can be characterized by mass spectrometry (MS)-based proteomics using customized protein databases generated from sets of transcripts previously detected by RNA-seq. This diversity has only been increased by the recent discovery that many translated alternative open reading frames rest unannotated at unsuspected locations of mRNAs and ncRNAs. These novel protein products, termed alternative proteins, have been left out of all previous custom database generation tools. Consequently, genetic variations that impact alternative open reading frames and variant peptides from their translated proteins are not detectable with current computational workflows. To fill this gap, we present OpenCustomDB, a bioinformatics tool that uses sample-specific RNaseq data to identify genomic variants in canonical and alternative open reading frames, allowing for more than one coding region per transcript. In a test reanalysis of a cohort of 16 patients with acute myeloid leukemia, 5666 peptides from alternative proteins were detected, including 201 variant peptides. We also observed that a significant fraction of peptide-spectrum matches previously assigned to peptides from canonical proteins got better scores when reassigned to peptides from alternative proteins. Custom protein libraries that include sample-specific sequence variations of all possible open reading frames are promising contributions to the development of proteomics and precision medicine. The raw and processed proteomics data presented in this study can be found in PRIDE repository with accession number PXD029240.

CrypticProteinDB: an integrated database of proteome and immunopeptidome derived non-canonical cancer proteins.

Translated non-canonical proteins derived from noncoding regions or alternative open reading frames (ORFs) can contribute to critical and diverse cellular processes. In the context of cancer, they also represent an under-appreciated source of targets for cancer immunotherapy through their tumor-enriched expression or by harboring somatic mutations that produce neoantigens. Here, we introduce the largest integration and proteogenomic analysis of novel peptides to assess the prevalence of non-canonical ORFs (ncORFs) in more than 900 patient proteomes and 26 immunopeptidome datasets across 14 cancer types. The integrative proteogenomic analysis of whole-cell proteomes and immunopeptidomes revealed peptide support for a nonredundant set of 9760 upstream, downstream, and out-of-frame ncORFs in protein coding genes and 12811 in noncoding RNAs. Notably, 6486 ncORFs were derived from differentially expressed genes and 340 were ubiquitously translated across eight or more cancers. The analysis also led to the discovery of thirty-four epitopes and eight neoantigens from non-canonical proteins in two cohorts as novel cancer immunotargets. Collectively, our analysis integrated both bottom-up proteogenomic and targeted peptide validation to illustrate the prevalence of translated non-canonical proteins in cancer and to provide a resource for the prioritization of novel proteins supported by proteomic, immunopeptidomic, genomic and transcriptomic data, available at https://www.maherlab.com/crypticproteindb.

Single-cell RNA sequencing reveals the suppressive effect of PPP1R15A inhibitor Sephin1 in antitumor immunity

Protein phosphatase 1 regulatory subunit 15A (PPP1R15A) is an important factor in the integrated stress response (ISR) in mammals and may play a crucial role in tumorigenesis. In our studies, we found an inhibitor of PPP1R15A, Sephin1, plays a protumorigenic role in mouse tumor models. By analyzing the single-cell transcriptome data of the mouse tumor models, we found that in C57BL/6 mice, Sephin1 treatment could lead to higher levels of ISR activity and lower levels of antitumor immune activities. Specifically, Sephin1 treatment caused reductions in antitumor immune cell types and lower expression levels of cytotoxicity-related genes. In addition, Tcell receptor (TCR) repertoire analysis demonstrated that the clonal expansion of tumor-specific Tcells was inhibited by Sephin1. A special TCR+ macrophage subtype in tumor was identified to be significantly depleted upon Sephin1 treatment, implying its key antitumor role. These results suggest that PPP1R15A has the potential to be an effective target for tumor therapy.

BONCAT-based Profiling of Nascent Small and Alternative Open Reading Frame-encoded Proteins.

RIBO-seq and proteogenomics have revealed that mammalian genomes harbor thousands of unannotated small and alternative open reading frames (smORFs, <100 amino acids, and alt-ORFs, >100 amino acids, respectively). Several dozen mammalian smORF-encoded proteins (SEPs) and alt-ORF-encoded proteins (alt-proteins) have been shown to play important biological roles, while the overwhelming majority of smORFs and alt-ORFs remain uncharacterized, particularly at the molecular level. Functional proteomics has the potential to reveal key properties of unannotated SEPs and alt-proteins in high throughput, and an approach to identify SEPs and alt-proteins undergoing regulated synthesis should be of broad utility. Here, we introduce a chemoproteomic pipeline based on bio-orthogonal non-canonical amino acid tagging (BONCAT) (Dieterich et al., 2006) to profile nascent SEPs and alt-proteins in human cells. This approach is able to identify cellular stress-induced and cell-cycle regulated SEPs and alt-proteins in cells. Graphical abstract Schematic overview of BONCAT-based chemoproteomic profiling of nascent, unannotated small and alternative open reading frame-encoded proteins (SEPs and alt-proteins).

Haploinsufficiency of EXT1 and Heparan Sulphate Deficiency Associated with Hereditary Multiple Exostoses in a Pakistani Family.

Background and Objectives: Hereditary multiple exostoses (HME) is a disease characterized by cartilage-capped bony protuberances at the site of growth plates of long bones. Functional mutations in the exostosin genes (EXT1 and EXT2) are reported to affect the hedgehog signalling pathways leading to multiple enchondromatosis. However, the exact role of each EXT protein in the regulation of heparan sulphate (HS) chain elongation is still an enigma. In this study, a Pakistani family with HME is investigated to find out the genetic basis of the disease. Materials and Methods: Genotyping of eight members of the family by amplifying microsatellite markers, tightly linked to the EXT1 and EXT2 genes. Results: The study revealed linkage of the HME family to the EXT1 locus 8q24.1. Sanger sequencing identified a heterozygous deletion (c.247Cdel) in exon 1 of EXT1, segregating with the disease phenotype in the family. In silico analysis predicted a shift in the frame causing an early stop codon (p.R83GfsX52). The predicted dwarf protein constituting 134 amino acids was functionally aberrant with a complete loss of the catalytic domain at the C-terminus. Interestingly, an alternative open reading frame 3 (ORF3) caused by the frame shift is predicted to encode a protein sequence, identical to the wild type and containing the catalytic domain, but lacking the first 100 amino acids of the wild-type EXT1 protein. Conclusion: Consequently, haploinsufficiency could be the cause of HME in the investigated family as the mutated copy of EXT1 is ineffective for EXT-1/2 complex formation. The predicted ORF3 protein could be of great significance in understanding several aspects of HME pathogenesis.

Transcriptomic complexity of the human malaria parasite Plasmodium falciparum revealed by long-read sequencing.

The Plasmodium falciparum human malaria parasite genome is incompletely annotated and does not accurately represent the transcriptomic diversity of this species. To address this need, we performed long-read transcriptomic sequencing. 5' capped mRNA was enriched from samples of total and nuclear-fractionated RNA from intra-erythrocytic stages and converted to cDNA library. The cDNA libraries were sequenced on PacBio and Nanopore long-read platforms. 12,495 novel isoforms were annotated from the data. Alternative 5' and 3' ends represent the majority of isoform events among the novel isoforms, with retained introns being the next most common event. The majority of alternative 5' ends correspond to genomic regions with features similar to those of the reference transcript 5' ends. However, a minority of alternative 5' ends showed markedly different features, including locations within protein-coding regions. Alternative 3' ends showed similar features to the reference transcript 3' ends, notably adenine-rich termination signals. Distinguishing features of retained introns could not be observed, except for a tendency towards shorter length and greater GC content compared with spliced introns. Expression of antisense and retained intron isoforms was detected at different intra-erythrocytic stages, suggesting developmental regulation of these isoform events. To gain insights into the possible functions of the novel isoforms, their protein-coding potential was assessed. Variants of P. falciparum proteins and novel proteins encoded by alternative open reading frames suggest that P. falciparum has a greater proteomic repertoire than the current annotation. We provide a catalog of annotated transcripts and encoded alternative proteins to support further studies on gene and protein regulation of this pathogen.

Analysis of nested alternate open reading frames and their encoded proteins.

Transcriptional and post-transcriptional mechanisms diversify the proteome beyond gene number, while maintaining a sequence relationship between original and altered proteins. A new mechanism breaks this paradigm, generating novel proteins by translating alternative open reading frames (Alt-ORFs) within canonical host mRNAs. Uniquely, ‘alt-proteins’ lack sequence homology with host ORF-derived proteins. We show global amino acid frequencies, and consequent biochemical characteristics of Alt-ORFs nested within host ORFs (nAlt-ORFs), are genetically-driven, and predicted by summation of frequencies of hundreds of encompassing host codon-pairs. Analysis of 101 human nAlt-ORFs of length ≥150 codons confirms the theoretical predictions, revealing an extraordinarily high median isoelectric point (pI) of 11.68, due to anomalous charged amino acid levels. Also, nAlt-ORF proteins exhibit a >2-fold preference for reading frame 2 versus 3, predicted mitochondrial and nuclear localization, and elevated codon adaptation index indicative of natural selection. Our results provide a theoretical and conceptual framework for exploration of these largely unannotated, but potentially significant, alternative ORFs and their encoded proteins.

An alternative CYB5A transcript is expressed in aneuploid ALL and enriched in relapse

BackgroundB-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a genetically heterogenous malignancy with poor prognosis in relapsed adult patients. The genetic basis for relapse in aneuploid subtypes such as near haploid (NH) and high hyperdiploid (HeH) BCP-ALL is only poorly understood. Pathogenic genetic alterations remain to be identified. To this end, we investigated the dynamics of genetic alterations in a matched initial diagnosis-relapse (ID-REL) BCP-ALL cohort. Here, we firstly report the identification of the novel genetic alteration CYB5Aalt, an alternative transcript of CYB5A, in two independent cohorts.MethodsWe identified CYB5alt in the RNAseq-analysis of a matched ID-REL BCP-ALL cohort with 50 patients and quantified its expression in various molecular BCP-ALL subtypes. Findings were validated in an independent cohort of 140 first diagnosis samples from adult BCP-ALL patients. Derived from patient material, the alternative open reading frame of CYB5Aalt was cloned (pCYB5Aalt) and pCYB5Aalt or the empty vector were stably overexpressed in NALM-6 cells. RNA sequencing was performed of pCYB5Aalt clones and empty vector controls followed by differential expression analysis, gene set enrichment analysis and complementing cell death and viability assays to determine functional implications of CYB5Aalt.ResultsRNAseq data analysis revealed non-canonical exon usage of CYB5Aalt starting from a previously undescribed transcription start site. CYB5Aalt expression was increased in relapsed BCP-ALL and its occurrence was specific towards the shared gene expression cluster of NH and HeH BCP-ALL in independent cohorts. Overexpression of pCYB5Aalt in NALM-6 cells induced a distinct transcriptional program compared to empty vector controls with downregulation of pathways related to reported functions of CYB5A wildtype. Interestingly, CYB5A wildtype expression was decreased in CYB5Aalt samples in silico and in vitro. Additionally, pCYB5Aalt NALM-6 elicited a more resistant drug response.ConclusionsAcross all age groups, CYB5Aalt was the most frequent secondary genetic event in relapsed NH and HeH BCP-ALL. In addition to its high subgroup specificity, CYB5Aalt is a novel candidate to be potentially implicated in therapy resistance in NH and HeH BCP-ALL. This is underlined by overexpressing CYB5Aalt providing first evidence for a functional role in BCL2-mediated apoptosis.

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation.

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

Mosaic translation hypothesis: chimeric polypeptides produced via multiple ribosomal frameshifting as a basis for adaptability.

How many different proteins can be produced from a single spliced transcript? Genome annotation projects overlook the coding potential of reading frames other than that of the reference open reading frames (refORFs). Recently, alternative open reading frames (altORFs) and their translational products, alternative proteins, have been shown to carry out important functions in various organisms. AltORFs overlapping refORFs or other altORFs in a different reading frame may be involved in one fundamental mechanism so far overlooked. A few years ago, it was proposed that altORFs may act as building blocks for chimeric (mosaic) polypeptides, which are produced via multiple ribosomal frameshifting events from a single mature transcript. We adopt terminology from that earlier discussion and call this mechanism mosaic translation. This way of extracting and combining genetic information may significantly increase proteome diversity. Thus, we hypothesize that this mechanism may have contributed to the flexibility and adaptability of organisms to a variety of environmental conditions. Specialized ribosomes acting as sensors probably played a central role in this process. Importantly, mosaic translation may be the main source of protein diversity in genomes that lack alternative splicing. The idea of mosaic translation is a testable hypothesis, although its direct demonstration is challenging. Should mosaic translation occur, we would currently highly underestimate the complexity of translation mechanisms and thus the proteome.