-
Cell Reports Oct 2018Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to...
Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information about ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-seq approaches lack the ability to distinguish between fragments protected by either ribosomes in active translation or inactive ribosomes. To overcome this possible limitation, we developed RiboLace, a method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied both in vitro and in vivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution.
Topics: Animals; Cell Line; High-Throughput Nucleotide Sequencing; Humans; Mice; Microspheres; Puromycin; RNA, Messenger; Ribosomes
PubMed: 30355487
DOI: 10.1016/j.celrep.2018.09.084 -
International Journal of Molecular... Nov 2019In 90% of pancreatic ductal adenocarcinoma cases, genetic alteration of the proto-oncogene Kras has occurred, leading to uncontrolled proliferation of cancerous cells....
In 90% of pancreatic ductal adenocarcinoma cases, genetic alteration of the proto-oncogene Kras has occurred, leading to uncontrolled proliferation of cancerous cells. Targeting Kras has proven to be difficult and the battle against pancreatic cancer is ongoing. A promising approach to combat cancer was the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, which can be used to genetically modify cells. To assess the potential of a CRISPR/CRISPR-associated protein 9 (Cas9) method to eliminate Kras mutations in cells, we aimed to knock-out the c.35G>A (p.G12D) Kras mutation. Therefore, three cell lines with a heterozygous Kras mutation (the human cell lines SUIT-2 and Panc-1 and the cell line TB32047 from a KPC mouse model) were used. After transfection, puromycin selection and single-cell cloning, proteins from two negative controls and five to seven clones were isolated to verify the knock-out and to analyze changes in key signal transduction proteins. Western blots showed a specific knock-out in the Kras protein, but wildtype Kras was expressed by all of the cells. Signal transduction analysis (for Erk, Akt, Stat3, AMPKα, and c-myc) revealed expression levels similar to the wildtype. The results described herein indicate that knocking-out the Kras mutation by CRISPR/Cas9 is possible. Additionally, under regular growth conditions, the knock-out clones resembled wildtype cells.
Topics: Alleles; Amino Acid Substitution; CRISPR-Cas Systems; Cell Line, Tumor; Gene Editing; Gene Expression Profiling; Gene Knockout Techniques; Gene Targeting; Humans; Mutation; Pancreatic Neoplasms; Proto-Oncogene Mas; Proto-Oncogene Proteins p21(ras); Signal Transduction
PubMed: 31739488
DOI: 10.3390/ijms20225706 -
Nature Communications Oct 2019Selectable markers are widely used in transgenesis and genome editing for selecting engineered cells with a desired genotype but the variety of markers is limited. Here...
Selectable markers are widely used in transgenesis and genome editing for selecting engineered cells with a desired genotype but the variety of markers is limited. Here we present split selectable markers that each allow for selection of multiple "unlinked" transgenes in the context of lentivirus-mediated transgenesis as well as CRISPR-Cas-mediated knock-ins. Split marker gene segments fused to protein splicing elements called "inteins" can be separately co-segregated with different transgenic vectors, and rejoin via protein trans-splicing to reconstitute a full-length marker protein in host cells receiving all intended vectors. Using a lentiviral system, we create and validate 2-split Hygromycin, Puromycin, Neomycin and Blasticidin resistance genes as well as mScarlet fluorescent proteins. By combining split points, we create 3- and 6-split Hygromycin resistance genes, demonstrating that higher-degree split markers can be generated by a "chaining" design. We adapt the split marker system for selecting biallelically engineered cells after CRISPR gene editing. Future engineering of split markers may allow selection of a higher number of genetic modifications in target cells.
Topics: CRISPR-Cas Systems; Cell Line, Tumor; Cinnamates; Drug Resistance, Bacterial; Gene Editing; Gene Transfer Techniques; Genetic Engineering; Genetic Vectors; HEK293 Cells; HeLa Cells; Humans; Hygromycin B; Induced Pluripotent Stem Cells; Inteins; Lentivirus; Luminescent Proteins; Neomycin; Nucleosides; Protein Splicing; Puromycin; Trans-Splicing; Transgenes
PubMed: 31672965
DOI: 10.1038/s41467-019-12891-2 -
Proceedings of the National Academy of... Dec 2019Mutations responsible for inherited disease may act by disrupting normal transcriptional splicing. Such mutations can be difficult to detect, and their effects difficult...
Mutations responsible for inherited disease may act by disrupting normal transcriptional splicing. Such mutations can be difficult to detect, and their effects difficult to characterize, because many lie deep within exons or introns where they may alter splice enhancers or silencers or introduce new splice acceptors or donors. Multiple mutation-specific and genome-wide approaches have been developed to evaluate these classes of mutations. We introduce a complementary experimental approach, cBROCA, which yields qualitative and quantitative assessments of the effects of genomic mutations on transcriptional splicing of tumor suppressor genes. cBROCA analysis is undertaken by deriving complementary DNA (cDNA) from puromycin-treated patient lymphoblasts, hybridizing the cDNA to the BROCA panel of tumor suppressor genes, and then multiplex sequencing to very high coverage. At each splice junction suggested by split sequencing reads, read depths of test and control samples are compared. Significant Z scores indicate altered transcripts, over and above naturally occurring minor transcripts, and comparisons of read depths indicate relative abundances of mutant and normal transcripts. BROCA analysis of genomic DNA suggested 120 rare mutations from 150 families with cancers of the breast, ovary, uterus, or colon, in >600 informative genotyped relatives. cBROCA analysis of their transcripts revealed a wide variety of consequences of abnormal splicing in tumor suppressor genes, including whole or partial exon skipping, exonification of intronic sequence, loss or gain of exonic and intronic splicing enhancers and silencers, complete intron retention, hypomorphic alleles, and combinations of these alterations. Combined with pedigree analysis, cBROCA sequencing contributes to understanding the clinical consequences of rare inherited mutations.
PubMed: 31843900
DOI: 10.1073/pnas.1915608116 -
Nature Communications Jul 2016The site-specific insertion of heterologous genetic material into genomes provides a powerful means to study gene function. Here we describe a modular system entitled...
The site-specific insertion of heterologous genetic material into genomes provides a powerful means to study gene function. Here we describe a modular system entitled CRISPaint (CRISPR-assisted insertion tagging) that allows precise and efficient integration of large heterologous DNA cassettes into eukaryotic genomes. CRISPaint makes use of the CRISPR-Cas9 system to introduce a double-strand break (DSB) at a user-defined genomic location. A universal donor DNA, optionally provided as minicircle DNA, is cleaved simultaneously to be integrated at the genomic DSB, while processing the donor plasmid at three possible positions allows flexible reading-frame selection. Applying this system allows to create C-terminal tag fusions of endogenously encoded proteins in human cells with high efficiencies. Knocking out known DSB repair components reveals that site-specific insertion is completely dependent on canonical NHEJ (DNA-PKcs, XLF and ligase-4). A large repertoire of modular donor vectors renders CRISPaint compatible with a wide array of applications.
Topics: CRISPR-Cas Systems; Gene Knock-In Techniques; Genetic Engineering; HEK293 Cells; Humans; Plasmids; Puromycin; Reading Frames
PubMed: 27465542
DOI: 10.1038/ncomms12338 -
Antibiotics (Basel, Switzerland) May 2016RNase P is an essential endonuclease in tRNA biogenesis, which generates the mature 5'-termini of tRNAs. Most forms of RNase P are ribonucleoproteins, i.e., they consist... (Review)
Review
RNase P is an essential endonuclease in tRNA biogenesis, which generates the mature 5'-termini of tRNAs. Most forms of RNase P are ribonucleoproteins, i.e., they consist of an essential RNA and protein subunits. The catalytic function of ribonucleoprotein RNase P enzymes resides entirely in the RNA subunit. Its high structural and functional diversity among representatives of a vast variety of phylogenetic domains indicates that RNase P could serve as a molecular target and a useful screening system for the development of new drugs in the battle against bacterial drug resistance.
PubMed: 27164152
DOI: 10.3390/antibiotics5020015 -
Critical Reviews in Biochemistry and... 2014In addition to the small and large ribosomal subunits, aminoacyl-tRNAs, and an mRNA, cellular protein synthesis is dependent on translation factors. The eukaryotic... (Review)
Review
In addition to the small and large ribosomal subunits, aminoacyl-tRNAs, and an mRNA, cellular protein synthesis is dependent on translation factors. The eukaryotic translation initiation factor 5A (eIF5A) and its bacterial ortholog elongation factor P (EF-P) were initially characterized based on their ability to stimulate methionyl-puromycin (Met-Pmn) synthesis, a model assay for protein synthesis; however, the function of these factors in cellular protein synthesis has been difficult to resolve. Interestingly, a conserved lysine residue in eIF5A is post-translationally modified to hypusine and the corresponding lysine residue in EF-P from at least some bacteria is modified by the addition of a β-lysine moiety. In this review, we provide a summary of recent data that have identified a novel role for the translation factor eIF5A and its hypusine modification in the elongation phase of protein synthesis and more specifically in stimulating the production of proteins containing runs of consecutive proline residues.
Topics: Amino Acid Sequence; Animals; Humans; Lysine; Models, Molecular; Molecular Sequence Data; Peptide Elongation Factors; Peptide Initiation Factors; Peptides; RNA-Binding Proteins; Ribosomes; Eukaryotic Translation Initiation Factor 5A
PubMed: 25029904
DOI: 10.3109/10409238.2014.939608 -
Computational and Structural... 2020Puromycin is a naturally occurring aminonucleoside antibiotic that inhibits protein synthesis by ribosome-catalyzed incorporation into the C-terminus of elongating... (Review)
Review
Puromycin is a naturally occurring aminonucleoside antibiotic that inhibits protein synthesis by ribosome-catalyzed incorporation into the C-terminus of elongating nascent chains, blocking further extension and resulting in premature termination of translation. It is most commonly known as a selection marker for cell lines genetically engineered to express a resistance transgene, but its additional uses as a probe for protein synthesis have proven invaluable across a wide variety of model systems, ranging from purified ribosomes and cell-free translation to intact cultured cells and whole animals. Puromycin is comprised of a nucleoside covalently bound to an amino acid, mimicking the 3' end of aminoacylated tRNAs that participate in delivery of amino acids to elongating ribosomes. Both moieties can tolerate some chemical substitutions and modifications without significant loss of activity, generating a diverse toolbox of puromycin-based reagents with added functionality, such as biotin for affinity purification or fluorophores for fluorescent microscopy detection. These reagents, as well as anti-puromycin antibodies, have played a pivotal role in advancing our understanding of the regulation and dysregulation of protein synthesis in normal and pathological processes, including immune response and neurological function. This manuscript reviews the current state of puromycin-based research, including structure and mechanism of action, relevant derivatives, use in advanced methodologies and some of the major insights generated using such techniques both in the lab and the clinic.
PubMed: 32435426
DOI: 10.1016/j.csbj.2020.04.014 -
Brain Research Bulletin Apr 2021Memory formation is a fundamental function of the nervous system that enables the experience-based adaptation of behaviour. The formation, recall and updating of... (Review)
Review
Memory formation is a fundamental function of the nervous system that enables the experience-based adaptation of behaviour. The formation, recall and updating of long-term memory (LTM) requires new protein synthesis through its direct involvement in neuronal processes, such as long-term potentiation (LTP), long-term depression (LTD) and synaptic scaling. We discuss the advantages and limitations of several emerging techniques which enable the tagging of newly synthesised proteins, including stable isotope labelling with amino acids in cell culture (SILAC), puromycin labelling, and non-canonical amino acid (NCAA) labelling. We further present how these methods allow for the identification and visualisation of proteins which are newly synthesised during different stages of memory formation. These emerging techniques will continue to expand our understanding of how memories are formed, consolidated and retrieved.
Topics: Animals; Brain; Hippocampus; Memory, Long-Term; Neuronal Plasticity; Proteomics; Synapses
PubMed: 33465403
DOI: 10.1016/j.brainresbull.2020.12.015 -
Nature Communications Apr 2020Here, we describe a drug-inducible genetic system for insect sex-separation that demonstrates proof-of-principle for positive sex selection in D. melanogaster. The...
Here, we describe a drug-inducible genetic system for insect sex-separation that demonstrates proof-of-principle for positive sex selection in D. melanogaster. The system exploits the toxicity of commonly used broad-spectrum antibiotics geneticin and puromycin to kill the non-rescued sex. Sex-specific rescue is achieved by inserting sex-specific introns into the coding sequences of antibiotic-resistance genes. When raised on geneticin-supplemented food, the sex-sorter line establishes 100% positive selection for female progeny, while the food supplemented with puromycin positively selects 100% male progeny. Since the described system exploits conserved sex-specific splicing mechanisms and reagents, it has the potential to be adaptable to other insect species of medical and agricultural importance.
Topics: Animals; Drosophila Proteins; Drosophila melanogaster; Drug Resistance; Exons; Female; Genetic Engineering; Genetics, Population; Gentamicins; Homozygote; Introns; Male; Pest Control; Puromycin; RNA Splicing; Sex Determination Analysis
PubMed: 32355156
DOI: 10.1038/s41467-020-16020-2