Papers by Hans E Johansson
eLife, 2020
Cell cycle is a cellular process that is subject to stringent control. In contrast to the
wealth ... more Cell cycle is a cellular process that is subject to stringent control. In contrast to the
wealth of knowledge of proteins controlling the cell cycle, very little is known about the molecular
role of lncRNAs (long noncoding RNAs) in cell-cycle progression. By performing genome-wide
transcriptome analyses in cell-cycle-synchronized cells, we observed cell-cycle phase-specific
induction of >2000 lncRNAs. Further, we demonstrate that an S-phase-upregulated lncRNA,
SUNO1, facilitates cell-cycle progression by promoting YAP1-mediated gene expression. SUNO1
facilitates the cell-cycle-specific transcription of WTIP, a positive regulator of YAP1, by promoting
the co-activator, DDX5-mediated stabilization of RNA polymerase II on chromatin. Finally, elevated
SUNO1 levels are associated with poor cancer prognosis and tumorigenicity, implying its pro-
survival role. Thus, we demonstrate the role of a S-phase up-regulated lncRNA in cell-cycle
progression via modulating the expression of genes controlling cell proliferation.
Molecular and Cellular Biology, Genetics
The multifaceted control of gene expression requires tight coordination of regulatory mechanisms ... more The multifaceted control of gene expression requires tight coordination of regulatory mechanisms at transcriptional and post-transcriptional level. Here, we studied the interdependence of transcription, splicing and polyadenylation events on single mRNA molecules by full-length mRNA sequencing. In MCF-7 breast cancer cells, we found 2,700 genes with interdependent alternative transcription, splicing and polyadenylation events, both in proximal and distant parts of mRNA molecules. The analysis of three human primary tissues revealed similar patterns of interdependency between transcription and mRNA processing events. We predict thousands of novel Open Reading Frames from the sequence of full-length mRNAs and obtained evidence for their translation by shotgun proteomics. The mapping database rescued 358 previously unassigned peptides and improved the assignment of others. By recognizing sample-specific amino-acid changes and novel splicing patterns, full-length mRNA sequencing improve...
Proceedings of the National Academy of Sciences of the United States of America, Feb 21, 2017
Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by progressive motor n... more Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by progressive motor neuron loss and caused by mutations in SMN1 (Survival Motor Neuron 1). The disease severity inversely correlates with the copy number of SMN2, a duplicated gene that is nearly identical to SMN1. We have delineated a mechanism of transcriptional regulation in the SMN2 locus. A previously uncharacterized long noncoding RNA (lncRNA), SMN-antisense 1 (SMN-AS1), represses SMN2 expression by recruiting the Polycomb Repressive Complex 2 (PRC2) to its locus. Chemically modified oligonucleotides that disrupt the interaction between SMN-AS1 and PRC2 inhibit the recruitment of PRC2 and increase SMN2 expression in primary neuronal cultures. Our approach comprises a gene-up-regulation technology that leverages interactions between lncRNA and PRC2. Our data provide proof-of-concept that this technology can be used to treat disease caused by epigenetic silencing of specific loci.
Methods in molecular biology (Clifton, N.J.), 2016
RNA fluorescence in situ hybridization (FISH), long an indispensable tool for the detection and l... more RNA fluorescence in situ hybridization (FISH), long an indispensable tool for the detection and localization of RNA, is becoming an increasingly important complement to other gene expression analysis methods. Especially important for long noncoding RNAs (lncRNAs), RNA FISH adds the ability to distinguish between primary and mature lncRNA transcripts and thus to segregate the site of synthesis from the site of action.We detail a streamlined RNA FISH protocol for the simultaneous imaging of multiple primary and mature mRNA and lncRNA gene products and RNA variants in fixed mammalian cells. The technique makes use of fluorescently pre-labeled, short DNA oligonucleotides (circa 20 nucleotides in length), pooled into sets of up to 48 individual probes. The overall binding of multiple oligonucleotides to the same RNA target results in fluorescent signals that reveal clusters of RNAs or single RNA molecules as punctate spots without the need for enzymatic signal amplification. Visualizatio...
Nucleic Acids Research, Dec 1, 1994
We describe a novel experimental approach to investigate mRNA translation. Antisense 2'-0-allyl o... more We describe a novel experimental approach to investigate mRNA translation. Antisense 2'-0-allyl oligoribonucleotides (oligos) efficiently arrest translation of targeted mRNAs in rabbit reticulocyte lysate and wheat germ extract while displaying minimal non-specific effects on translation. Oligo/mRNAhybrids positioned anywhere within the 5' UTR or the first-20 nucleotides of the open reading frame block cap-dependent translation initiation with high specificity. The thermodynamic stability of hybrids between 2'-0-alkyl oligos and RNA permits translational inhibition with oligos as short as 10 nucleotides. This inhibition is independent of RNase H cleavage or modifications which render the mRNA untranslatable. We show that 2'-0-alkyl oligos can also be employed to interfere with cap-independent internal initiation of translation and to arrest translation elongation. The latter is accomplished by UV-crosslinking of psoralentagged 2'-0-methyloligoribonucleotides to the mRNA within the open reading frame. The utility of 2'-0alkyloligoribonucleotides to arrest translation from defined positions within an mRNA provides new approaches to investigate mRNA translation.
Cloning and expression The BTI1 azoreductase clone SO1-2 was isolated from a local soil sample by... more Cloning and expression The BTI1 azoreductase clone SO1-2 was isolated from a local soil sample by use of degenerate PCR. The azoreductase (C70V) open reading frame (GenBank ID: EU664999) was codon-optimized for expression in E. coli and mammalian cells and equipped with peptide tags for immunorecognition (myc) and affinity purification (His 6 ). Tagged BTI1 (C70V) was expressed and purified making use of the C-term His 6 tag from E. coli BL21(DE3) pLysS essentially as recommended (Novagen). Routinely, 50 ml cultures yielded 2-5 mg of >97 % pure protein. Gel-electrophoresis Samples for SDS-PAGE were denatured in Laemmli loading buffer and separated on a 14% Tris-Gly gel (Invitrogen). Samples in Tris-Cl glycerol were separated on a 14 % native polyacrylamide gel (Invitrogen). Proteins in the gels were stained with GelCode (Pierce). The size markers were: PAGE-Ruler (Fermentas), Native-Mark (Invitrogen). HPLC and Mass Spectrometry The absorption spectra and the elution profiles in anion exchange HPLC of native and denatured BTI1(C70V) as well as of standard flavin co-factors were recorded on an HP diode array spectrometer and a Waters HPLC. BTI1(C70V) was subjected to ESI-MS (Waters) to determine the mass of the denatured polypeptide and that of the yellow non-covalently bound cofactor. Pro-fluorescent substrate synthesis and purification Pro-fluorescent probes were synthesized on a Biosearch 8700 DNA synthesizer using standard phosphoramidite reagents. Samples were purified by AX-HPLX, and salts removed on a reverse phase cartridge. Purity was assessed by RP-and AX-HPLC. The absence of absorption from free amines (quencher degradation products) confirmed dye integrity. Quantitative azoreduction All buffers were at 200 mM and NADPH at 200 µM. In both absorbance and fluorescence assays, 200 µl samples were placed in 96-well plates. Typically 0.5-2.0 µg of enzyme was used per reaction. Apparent K M and V max values were determined by varying the concentration of azo dye substrates (0.5-50 µM) and making Lineweaver-Burk plots. Reaction rates were calculated by fitting the initial part of the absorbance decay by linear regression to find the slope. Absorption spectra were recorded in a HP 8453 diode array spectrophotometer, and absorbance measurements were recorded in a Tecan Safire plate reader.
The Journal of biological chemistry, Jan 15, 1993
Hemoglobin synthesis in red cells is the major iron utilization pathway in the human body and acc... more Hemoglobin synthesis in red cells is the major iron utilization pathway in the human body and accounts for > 80% of systemic iron turnover. The first step in erythroid heme biosynthesis is catalyzed by a tissue-specific isoform of 5-aminolevulinate synthase (ALAS). The previous identification of iron-responsive elements in the 5'-untranslated region of human and murine erythroid ALAS mRNA raised the intriguing possibility that eALAS expression might be under iron-dependent translational control. As a consequence, a single post-transcriptional regulatory system could coordinate cellular iron acquisition via the transferrin receptor, storage via ferritin, and utilization via eALAS. We directly demonstrate iron-dependent translational regulation of eALAS mRNA in murine erythroleukemia (MEL) cells. The iron-responsive element motif contained in eALAS mRNA is shown to be sufficient to confer translational control to a reporter mRNA both in transfected MEL cells and in vitro.
Progress in nucleic acid research and molecular biology, 1994
ABSTRACT
Insect Biochemistry, 1990
Last instar larvae of Trichoplusia ni respond to injections of bacteria with a set of inducible i... more Last instar larvae of Trichoplusia ni respond to injections of bacteria with a set of inducible immune proteins. The induced proteins are related to the known immune proteins attacins, ceeropins and lysozyme from Hyalophora cecropia. The antibacterial response was elicited most effectively by bacteria. An injection of Autographa californica nuclear polyhedrosis virus did not cause an induction any larger than that caused by an injection of Ringer's saline. No antibacterial response was observed at all when the virus was given orally. Immune hemolymph was fractionated electrophoretically and the gel probed both with a bacterial overlay and Western blotting. There were three to four antibacterial bands, some of which cross-reacted with anti-Hyalophora cecropin antibodies. In SDS-Western blots one band had the same mobility as attacin. Isoelectric focusing gels indicate multiple forms of attacin from T. ni. Lysozyme activity was also shown to be induced. A suppression of the hemolymph's ability to melanize was noted in larvae injected with baculovirus or bacteria and to a lesser extent with Ringer's solution. The virus titer in the bemolymph increased similarly, whether the immune system had been induced with bacteria or not. The viral infection also caused some breakdown of many hemolymph proteins including the antibacterial proteins.
Methods Mol Biol, 2014
RNA fluorescence in situ hybridization (FISH) has long been an indispensable tool for the detecti... more RNA fluorescence in situ hybridization (FISH) has long been an indispensable tool for the detection and localization of RNA and is increasingly becoming an important complement to other gene expression analysis methods. We detail a streamlined RNA FISH protocol for the simultaneous imaging of multiple RNA gene products and RNA variants in fixed mammalian cells. The technique utilizes fluorescently pre-labeled, short DNA oligonucleotides (20 nucleotides in length), pooled into sets of up to 48 individual probes. The overall binding of multiple oligonucleotides to the same RNA target results in punctate fluorescent signals representing individual RNA molecules without the need for enzymatic signal amplification. Visualization of these punctate signals, through the use of wide-field fluorescence microscopy, enables the quantification of single RNA transcripts. Additionally, by utilizing probe sets with spectrally distinct fluorophores, multiplex analysis of specific RNAs, or RNA variants, can be achieved. We focus on the detection of a cytoplasmic mRNA and a nuclear long noncoding RNA to illustrate the benefits of this method for cell-specific detection and subcellular localization. Post-processing of images and spot counting is briefly discussed to demonstrate the capabilities of this method for the statistical analysis of RNA molecule number per cell, which is information that can be utilized to determine overall gene expression levels and cell-to-cell gene expression variation.
Proceedings of the National Academy of Sciences, 1998
Most mutations in the sequence of the RNA hairpin that specifically binds MS2 coat protein either... more Most mutations in the sequence of the RNA hairpin that specifically binds MS2 coat protein either reduce the binding affinity or have no effect. However, one RNA mutation, a uracil to cytosine change in the loop, has the unusual property of increasing the binding affinity to the protein by nearly 100-fold. Guided by the structure of the protein–RNA complex, we used a series of protein mutations and RNA modifications to evaluate the thermodynamic basis for the improved affinity: The tight binding of the cytosine mutation is due to (i) the amino group of the cytosine residue making an intra-RNA hydrogen bond that increases the propensity of the free RNA to adopt the structure seen in the complex and (ii) the increased affinity of hydrogen bonds between the protein and a phosphate two bases away from the cytosine residue. The data are in good agreement with a recent comparison of the cocrystal structures of the two complexes, where small differences in the two structures are seen at th...
Nucleic Acids Research, 2000
Genomic SELEX is a method for studying the network of nucleic acid-protein interactions within an... more Genomic SELEX is a method for studying the network of nucleic acid-protein interactions within any organism. Here we report the discovery of several interesting and potentially biologically important interactions using genomic SELEX. We have found that bacteriophage MS2 coat protein binds several Escherichia coli mRNA fragments more tightly than it binds the natural, well-studied, phage mRNA site. MS2 coat protein binds mRNA fragments from rffG (involved in formation of lipopolysaccharide in the bacterial outer membrane), ebgR (lactose utilization repressor), as well as from several other genes. Genomic SELEX may yield experimentally induced artifacts, such as molecules in which the fixed sequences participate in binding. We describe several methods (annealing of oligonucleotides complementary to fixed sequences or switching fixed sequences) to eliminate some, or almost all, of these artifacts. Such methods may be useful tools for both randomized sequence SELEX and genomic SELEX.
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Papers by Hans E Johansson
wealth of knowledge of proteins controlling the cell cycle, very little is known about the molecular
role of lncRNAs (long noncoding RNAs) in cell-cycle progression. By performing genome-wide
transcriptome analyses in cell-cycle-synchronized cells, we observed cell-cycle phase-specific
induction of >2000 lncRNAs. Further, we demonstrate that an S-phase-upregulated lncRNA,
SUNO1, facilitates cell-cycle progression by promoting YAP1-mediated gene expression. SUNO1
facilitates the cell-cycle-specific transcription of WTIP, a positive regulator of YAP1, by promoting
the co-activator, DDX5-mediated stabilization of RNA polymerase II on chromatin. Finally, elevated
SUNO1 levels are associated with poor cancer prognosis and tumorigenicity, implying its pro-
survival role. Thus, we demonstrate the role of a S-phase up-regulated lncRNA in cell-cycle
progression via modulating the expression of genes controlling cell proliferation.
wealth of knowledge of proteins controlling the cell cycle, very little is known about the molecular
role of lncRNAs (long noncoding RNAs) in cell-cycle progression. By performing genome-wide
transcriptome analyses in cell-cycle-synchronized cells, we observed cell-cycle phase-specific
induction of >2000 lncRNAs. Further, we demonstrate that an S-phase-upregulated lncRNA,
SUNO1, facilitates cell-cycle progression by promoting YAP1-mediated gene expression. SUNO1
facilitates the cell-cycle-specific transcription of WTIP, a positive regulator of YAP1, by promoting
the co-activator, DDX5-mediated stabilization of RNA polymerase II on chromatin. Finally, elevated
SUNO1 levels are associated with poor cancer prognosis and tumorigenicity, implying its pro-
survival role. Thus, we demonstrate the role of a S-phase up-regulated lncRNA in cell-cycle
progression via modulating the expression of genes controlling cell proliferation.