| 1. |
- Huch, Susanne, 1981-
(författare)
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Spatial control of mRNA stability in yeast
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The degradation of mRNA is an important modulator of gene expression and the ultimate fate of messenger mRNA. Important steps in the degradation of mRNA include initial shortening of its poly(A) tail followed by the subsequent removal of the m7G cap. These two processes are linked temporally as well as spatially. In addition to physical interactions between proteins involved in these two processes, deadenylation and decapping enzymes and accessory factors are found in P bodies. P bodies are aggregates of protein and mRNA that are induced upon stress in all eukaryotes examined. In this thesis, I examine the spatial localization of decapping factors and explore the role of P bodies in mRNA turnover in the yeast Saccharomyces cerevisiae. This thesis is based on three underlying principles. First, mRNA decapping factors are membrane associated. More so, we show that decapping factors can be co-localized with the endoplasmic reticulum and Golgi apparatus. Second, although P bodies were proposed as sites of mRNA decay, we found that they stabilize mRNA. We examined the role of P bodies in mRNA turnover using a mutant defective in their assembly, edc3∆ lsm4∆C. This strain is mutated in two decapping activators. It combines a deletion of the gene encoding the Edc3 protein and lacks the prion-like domain of Lsm4. Using the edc3∆ lsm4∆C mutant, we demonstrate that mRNA stability is significantly reduced in the absence of P bodies for longer-lived mRNA. The effect of mRNA destabilization was due to increased deadenylation and decapping dependence. Finally, the decapping factor usually found in the cytoplasm, but accumulates in the nucleus in the P body deficient strain (edc3∆ lsm4∆C). This implies a possible role in modulating transcription.A model for the functioning of P bodies that is consistent with our work is that P bodies serve a role as a cytoplasmic sink for degradation factors. By regulating the access of the cytosol to proteins involved in mRNA turnover, P bodies can modulate mRNA stability. This suggests a role for P bodies under stress and their potential importance in stress adaptation.
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| 2. |
- Liljeruhm, Josefine, et al.
(författare)
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Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology
- 2018
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Ingår i: Journal of Biological Engineering. - : BIOMED CENTRAL LTD. - 1754-1611. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Background: Coral reefs are colored by eukaryotic chromoproteins (CPs) that are homologous to green fluorescent protein. CPs differ from fluorescent proteins (FPs) by intensely absorbing visible light to give strong colors in ambient light. This endows CPs with certain advantages over FPs, such as instrument-free detection uncomplicated by ultra-violet light damage or background fluorescence, efficient Forster resonance energy transfer (FRET) quenching, and photoacoustic imaging. Thus, CPs have found utility as genetic markers and in teaching, and are attractive for potential cell biosensor applications in the field. Most near-term applications of CPs require expression in a different domain of life: bacteria. However, it is unclear which of the eukaryotic CP genes might be suitable and how best to assay them.Results: Here, taking advantage of codon optimization programs in 12 cases, we engineered 14 CP sequences (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, mefiBlue, aeBlue, amilCP, tsPurple and gfasPurple) into a palette of Escherichia coil BioBrick plasmids. BioBricks comply with synthetic biology's most widely used, simplified, cloning standard. Differences in color intensities, maturation times and fitness costs of expression were compared under the same conditions, and visible readout of gene expression was quantitated. A surprisingly large variation in cellular fitness costs was found, resulting in loss of color in some overnight liquid cultures of certain high-copy-plasmid-borne CPs, and cautioning the use of multiple CPs as markers in competition assays. We solved these two problems by integrating pairs of these genes into the chromosome and by engineering versions of the same CP with very different colors.Conclusion: Availability of 14 engineered CP genes compared in E coil, together with chromosomal mutants suitable for competition assays, should simplify and expand CP study and applications. There was no single plasmid-borne CP that combined all of the most desirable features of intense color, fast maturation and low fitness cost, so this study should help direct future engineering efforts.
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| 3. |
- Thuresson, Ann-Charlotte, et al.
(författare)
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Multiple forms of poly(A) polymerases in human cells
- 1994
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 91:3, s. 979-983
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Tidskriftsartikel (refereegranskat)abstract
- We have cloned human poly(A) polymerase (PAP) mRNA as cDNA in Escherichia coli. The primary structure of the mRNA was determined and compared to the bovine PAP mRNA sequence. The two sequences were 97% identical at the nucleotide level, which translated into 99% similarity at the amino acid level. Polypeptides representing recombinant PAP were expressed in E. coli, purified, and used as antigens to generate monoclonal antibodies. Western blot analysis using these monoclonal antibodies as probes revealed three PAPs, having estimated molecular masses of 90, 100, and 106 kDa in HeLa cell extracts. Fractionation of HeLa cells showed that the 90-kDa polypeptide was nuclear while the 100- and 106-kDa species were present in both nuclear and cytoplasmic fractions. The 106-kDa PAP was most likely a phosphorylated derivative of the 100-kDa species. PAP activity was recovered in vitro by using purified recombinant human PAP. Subsequent mutational analysis revealed that both the N- and C-terminal regions of PAP were important for activity and suggested that cleavage and polyadenylylation specificity factor (CPSF) interacted with the C-terminal region of PAP. Interestingly, tentative phosphorylation sites have been identified in this region, suggesting that phosphorylation/dephosphorylation may regulate the interaction between the two polyadenylylation factors PAP and CPSF.
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| 4. |
- Åström, Anders, et al.
(författare)
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A simple procedure for isolation of eukaryotic mRNA polyadenylation factors
- 1991
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 202:3, s. 765-773
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Tidskriftsartikel (refereegranskat)abstract
- We have devised a simple chromatographic procedure which isolates five polyadenylation factors that are required for polyadenylation of eukaryotic mRNA. The factors were separated from each other by fractionation of HeLa cell nuclear extract in two consecutive chromatographic steps. RNA cleavage at the L3 polyadenylation site of human adenovirus 2 required at least four factors. Addition of adenosine residues required only two of these factors. The fractionation procedure separates two components that are both likely to be poly(A) polymerases. The candidate poly(A) polymerases were interchangeable and participated during both RNA cleavage and adenosine addition. They were discriminated from each other by chromatographic properties, heat sensitivity and divalent cation requirement. We have compared our data with published information and have been able to correlate the activities that we have isolated to previously identified polyadenylation factors. However, we have not been able to assign one of the candidate poly(A) polymerases to a previously identified poly(A) polymerase. This simple fractionation procedure can be used for generating an in vitro reconstituted system for polyadenylation within a short period of time.
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| 5. |
- Åström, Jonas, et al.
(författare)
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In vitro deadenylation of mammalian mRNA by a HeLa cell 3' exonuclease
- 1991
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Ingår i: EMBO Journal. - 0261-4189 .- 1460-2075. ; 10:10, s. 3067-3071
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Tidskriftsartikel (refereegranskat)abstract
- We have identified a 3' exonuclease in HeLa cell extracts which deadenylates mammalian mRNA and leaves the mRNA body intact after poly(A) removal. Only homopolymeric adenosine tails located at the 3' end were efficiently removed by the exonuclease. The poly(A) removing activity did not require any specific sequences in the mRNA body either for poly(A) removal or for accumulation of the deadenylated mRNA. We conclude that the poly(A) removing activity is a 3' exonuclease since (i) reaction intermediates gradually lose the poly(A) tail, (ii) degradation is prevented by the presence of a cordycepin residue at the 3' end and (iii) RNAs having internally located poly(A) stretches are poor substrates for degradation. The possible involvement of the poly(A) removing enzyme in regulating mRNA translation and stability is discussed.
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| 6. |
- Åström, Jonas, et al.
(författare)
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Properties of a HeLa cell 3' exonuclease specific for degrading poly(A) tails of mammalian mRNA.
- 1992
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 267:25, s. 18154-18159
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Tidskriftsartikel (refereegranskat)abstract
- A HeLa cell 3'-exonuclease with properties of a mammalian mRNA poly(A) tail-removing enzyme has been characterized. The exonuclease shows high specificity for the poly(A) tail, and it is single strand-specific and requires a 3'-hydroxyl group for its activity. During degradation 5'-AMP is liberated as a product, and a 3'-OH group is left on the last adenosine residue of the remaining poly(A) tail. The activity is inhibited by 5'-AMP and can be competed by poly(A)-containing mRNA or poly(A). Based on these findings we propose a reaction pathway for poly(A) tail removal catalyzed by the HeLa cell poly(A) tail-specific 3' exonuclease.
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| 7. |
- Alkemar, Gunnar, 1970-
(författare)
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Ribosome and ribosomal RNA Structure : An experimental and computational analysis of expansion segments in eukaryotic ribosomal RNA
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ribosomes are large ribonucleoprotein complexes which incorporate amino acids into peptide chains during translational process in all types of living cells. The eukaryotic ribosome is larger compared to its prokaryotic counterpart. The size differences are due to a larger protein part and that the rRNA contains eukaryote specific expansion segments (ES). Cryo-EM reconstruction has visualized many ES on the ribosomal surface which have given clues about function and structural features. However, the secondary structures of most ES are unknown or ill defined. In this thesis, the secondary and also to a certain extent the tertiary structures of several ES are determined by using computational methods and biochemical experimental techniques. The juxtaposition of ES6 close to ES3 in the Cryo-EM image of the yeast ribosome suggested that ES3 and ES6 might interact. A computational analysis of more than 2900 sequences shows that a complementary helical region of seven to nine contiguous base pairs can form between ES3 and ES6 in almost all analyzed sequences. Biochemical in situ experiments support the proposed interaction. Secondary structure models are presented for ES3 and ES6 in 18S rRNA and ES39 in 28S rRNA, where homologous structural elements could be modeled in the experimentally analyzed ribosomes from fungi, plants and mammals. The structure models were further supported by computational methods where the ES6 structure and the ES39 structure could be formed in more than 6000 and 900 sequences respectively. A tertiary structure model of ES3 and ES6 including the helical interaction is presented. An in vitro transcribed and folded ES6 sequence differed from that observed in situ, suggesting that chaperones, ribosomal proteins, and/or the tertiary rRNA interaction could be involved in the in vivo folding of ES6. An analysis of the similarities between ES39 structures suggests that it might be under selective constraint to preserve its secondary structure.
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| 8. |
- Alping, Peter, et al.
(författare)
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Validation of the Swedish Multiple Sclerosis Register Further Improving a Resource for Pharmacoepidemiologic Evaluations
- 2019
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Ingår i: Epidemiology. - : Lippincott Williams & Wilkins. - 1044-3983 .- 1531-5487. ; 30:2, s. 230-233
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Tidskriftsartikel (refereegranskat)abstract
- The Swedish Multiple Sclerosis Register is a national register monitoring treatment and clinical course for all Swedish multiple sclerosis (MS) patients, with high coverage and close integration with the clinic. Despite its great value for epidemiologic research, it has not previously been validated. In this brief report, we summarize a large validation of >3,000 patients in the register using clinical chart review in the context of the COMBAT-MS study. While further improving the data quality for a central cohort of patients available for future epidemiologic research, this study also allowed us to estimate the accuracy and completeness of the register data.
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| 9. |
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| 10. |
- Ban, Ilija, et al.
(författare)
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Overtreatment of displaced midshaft clavicle fractures : A survey of hospitals in Sweden, Denmark, and Finland
- 2016
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Ingår i: Acta Orthopaedica. - : Medical Journals Sweden AB. - 1745-3674 .- 1745-3682. ; 87:6, s. 541-545
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Tidskriftsartikel (refereegranskat)abstract
- Background and purpose The best treatment for displaced clavicle fractures has been debated for decades. Operative treatment has become more common. However, several randomized trials comparing non-operative and operative treatment have not shown any compelling evidence in favor of surgery. We identified the preferred treatment of displaced midshaft clavicle fractures at public hospitals in 3 countries in Scandinavia.Patients and methods A purpose-made multiple-choice questionnaire in English was sent to all public hospitals in Denmark, Sweden, and Finland. This was addressed to the orthopedic surgeon responsible for treatment of clavicle fractures, and completed questionnaires were obtained from 85 of 118 hospitals.Results In the 3 countries, 69 of the 85 hospitals that responded would treat displaced clavicle fractures operatively. Clear criteria for treatment allocation were used at 58 of the hospitals, with the remaining 27 using individual assessment in collaboration with the patient. Precontoured locking plates were mostly used, placed either superiorly (64/85) or anteriorly (10/85).Interpretation Displaced midshaft clavicle fractures are mainly treated operatively in Sweden, Denmark, and Finland. This treatment is not supported by compelling evidence.
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