A Computational Approach to Search for Non-Coding RNAs in Large Genomic Data

Gräf, Stefan; Teune, Jan-Hendrik; Strothmann, Dirk; Kurtz, Stefan; Steger, Gerhard

doi:10.1007/3-540-28130-4_3

Cited by 2 publications

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“…To address the question of differing viral protein translation efficiencies, we also examined the expression of two additional structural proteins, VP2 and VP6 (Figures S7 and S8). Our selection of these two proteins was due to the high copy number required for RV particle formation, VP6 is known to be the most abundantly expressed structural protein [54] and has a segment specific enhancer in addition to the RV specific 3′ translational recognition sequence [55], [56]. We concluded that neither unmodified nor polyadenylated RV ssRNAs, obtained from either in vitro transcribed cDNAs or from DLPs, can act as efficient templates for protein synthesis when transfected into cells and therefore, by definition, were not autonomously infectious.…”

Section: Resultsmentioning

confidence: 99%

Experimental Pathways towards Developing a Rotavirus Reverse Genetics System: Synthetic Full Length Rotavirus ssRNAs Are Neither Infectious nor Translated in Permissive Cells

2013

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At present the ability to create rationally engineered mutant rotaviruses is limited because of the lack of a tractable helper virus-free reverse genetics system. Using the cell culture adapted bovine RV RF strain (G6P6 [1]), we have attempted to recover infectious RV by co-transfecting in vitro transcribed ssRNAs which are identical in sequence to the positive sense strand of each of the 11 dsRNA genomic segments of the RF strain. The RNAs were produced either from cDNAs cloned by a target sequence-independent procedure, or from purified double layered RV particles (DLPs). We have validated their translational function by in vitro synthesis of 35S-labelled proteins in rabbit reticulocyte lysates; all 11 proteins encoded by the RV genome were expressed. Transfection experiments with DLP- or cDNA-derived ssRNAs suggested that the RNAs do not act independently as mRNAs for protein synthesis, once delivered into various mammalian cell lines, and exhibit cytotoxicity. Transfected RNAs were not infectious since a viral cytopathic effect was not observed after infection of MA104 cells with lysates from transfected cells. By contrast, an engineered mRNA encoding eGFP was expressed when transfected under identical conditions into the same cell lines. Co-expression of plasmids encoding NSP2 and NSP5 using a fowlpox T7 polymerase recombinant virus revealed viroplasm-like structure formation, but this did not enable the translation of transfected RV ssRNAs. Attempts to recover RV from ssRNAs transcribed intracellularly from transfected cDNAs were also unsuccessful and suggested that these RNAs were also not translated, in contrast to successful translation from a transfected cDNA encoding an eGFP mRNA.

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Section: Resultsmentioning

confidence: 99%

Experimental Pathways towards Developing a Rotavirus Reverse Genetics System: Synthetic Full Length Rotavirus ssRNAs Are Neither Infectious nor Translated in Permissive Cells

2013

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“…With help of the preliminary consensus structure, creation of either a pattern or a covariance model (CM) is possible. Both allow to search–for patterns with programs like PatScan [ 61 ] or HyPa [ 1 ] and for CMs with programs like infernal [ 62 ] or RSEARCH [ 2 ]–more specifically for further members of the RNA group under inspection.…”

Section: Discussionmentioning

confidence: 99%

“…Prediction of RNA structure as well as searches for homologues in large genomic sequence databases play a prominent role in the era of non-coding RNAs (ncRNAs). Structure prediction may provide insight into RNA function, and pattern-based database searches [ 1 , 2 ] may reveal new homologues, without the need for time-consuming experiments. Prerequisite for these predictions and searches as well as for inference of phylogeny [ 3 - 5 ] is the existence of an alignment of RNA homologues correct in terms of both sequence and structure.…”

Section: Introductionmentioning

confidence: 99%

ConStruct: Improved construction of RNA consensus structures

2008

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Background: Aligning homologous non-coding RNAs (ncRNAs) correctly in terms of sequence and structure is an unresolved problem, due to both mathematical complexity and imperfect scoring functions. High quality alignments, however, are a prerequisite for most consensus structure prediction approaches, homology searches, and tools for phylogeny inference. Automatically created ncRNA alignments often need manual corrections, yet this manual refinement is tedious and error-prone.

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A Computational Approach to Search for Non-Coding RNAs in Large Genomic Data

Cited by 2 publications

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Experimental Pathways towards Developing a Rotavirus Reverse Genetics System: Synthetic Full Length Rotavirus ssRNAs Are Neither Infectious nor Translated in Permissive Cells

Experimental Pathways towards Developing a Rotavirus Reverse Genetics System: Synthetic Full Length Rotavirus ssRNAs Are Neither Infectious nor Translated in Permissive Cells

ConStruct: Improved construction of RNA consensus structures

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