Biology subjects

Rauwerda, H.

Publications and source records attributed to Rauwerda, H..

8 recordsLinked to original sources

bioRxiv2025

Extreme GC3 Codon Bias in a Novel Brown Seaweed Virus Results in Pseudoambigrammatic Characteristics

Although viruses are often studied in relation to disease, they can also offer valuable insights into RNA functionality. Due to their vast range of hosts and high mutation rates, viruses can quickly adapt to changing environments. It is therefore expected that viruses exploit all possible RNA functionalities to develop strategies for host infection and propagation. Our aim is to discover RNA viruses with survival strategies that reveal unknown RNA characteristics and functionalities. We have chosen seaweed as host organism because relatively little is known about RNA viruses in seaweed. Additionally, seaweeds are evolutionary distant from terrestrial plants and inhabit environments with entirely different conditions, increasing the likelihood of discovering new RNA viruses with unknown survival strategies. Here we report a new, unclassified negative-stranded single-strand RNA virus from seaweed Saccharina latissima that showed a unique genome characteristic. The novel virus, named SalaUV-NL1, contains a large segment of 7.065 nucleotides coding for a 2.296 amino acids protein and a small segment of 2.120 nucleotides coding for a 328 plus a potential 315+ amino acids protein. The large segment also appeared to have an additional large open reading frame (ORF) on the negative strand, suggesting it could be an ambigrammatic virus. However, the virus displayed an extreme GC3 codon bias (98%) in the (+)ORF, which differs from the typical avoidance of just the reverse-complement stop codons seen in ambigrammatic viruses. Consequently, the (-)ORF appears to occur accidental and is unlikely to encode a functional protein. Therefore, we have termed our virus to be pseudoambigrammatic. Several other viruses with the same pseudogrammatic high GC3 codon bias were identified in Genbank, most without a (-)ORF. The SalaUV-NL1 virus codon bias surpasses the high GC3 codon bias of the S. latissima host genes, however other species that host pseudoambigrammatic viruses do not display a high GC3 codon bias. Comparison of several variants of SalaUV-NL1 showed that the relative nucleotide change rate, particularly for silent changes from A/T to G/C, was extremely high (up to 81%). This novel virus demonstrates a new survival strategy of RNA viruses that is not yet understood.

genomics↗

bioRxiv2024

Discovery of new and variant Potyviruses in Asparagales plants from a Dutch urban botanic garden

Although viruses play an important role in human health and plant health, most viruses are still undetected. One of the reasons is that they may evolve in distinct natural habitats that are not yet extensively sampled for virus analysis. An example of such a habitat is urban botanic gardens. We analyzed 25 Asparagales plants with a mild to severe disease phenotype from a Dutch urban botanic garden for the presence of known an unknown virus types and virus variants by smallRNA-seq and RNA-seq. We found in all samples evidence for (past) Potyviridae presence, mostly Ornithogalum virus (OV) and Ornithogalum mosaic virus (OrMV), as well as a new Iris mild mosaic virus variant (IMMV) and a yet unknown species of Potyvirus. Also, presence of a new Betaflexivirus, a new Polerovirus and a new Phenuivirus were detected. Most analyzed plants harbored multiple viruses, 18 out of 25 plants showed evidence for three to seven viruses and 12 out of 13 viruses were present in four to 11 samples. In this study, we describe the characteristics of a newly discovered Potyvirus and identify several variants of known potyviruses. We place these findings in the context of known viruses. However, we were unable to link these potyviruses to any specific disease phenotype.

genomics↗

bioRxiv2022

Cellular Factors Involved in Transcriptome Dynamics in Early Zebrafish Embryogenesis

At gastrulation in the zebrafish embryogenesis, the embryonic genome is switched on to produce transcripts that are used for the maintenance and development of the embryo. In a previous study from late blastula to mid gastrula on the transcriptomes of 179 individual embryos, we capture the transcriptome dynamics via ten gene-expression types. Here we study the factors that regulate these transcriptome dynamics by in extensive silico analyses and two small-RNA sequencing experiments. We analyzed mechanisms that would make it possible for the embryo to achieve the tight regulation of gene expression that was observed, not only during development, but also when individual embryos were compared. We found that many of the gene-expression regulatory factors that are available to the embryo are operational in the different gene-expression types and act concurrently with not one mechanism prevailing in this developmental phase. We also saw that at least one of the regulatory mechanisms, the expression of members of the miRNA-430 family again is very tightly regulated, both during development as well as when miRNA expression from individual embryos is compared.

developmental biology↗

bioRxiv2020

Overhauling a faulty control in the CDC-recommended SARS-CoV-2 RT-PCR test

To battle the COVID-19 pandemic, widespread testing for the presence of the SARS-CoV-2 virus is worldwide being employed by specific real-time RT-PCR (rRT-PCR) of viral RNA. The CDC has issued a recommended panel of PCR-based test sets that entail several primer/probe sets that target the SARS-CoV-2 N-gene, but also one that targets the human RNase P gene (h-RP) as a positive control for RNA extraction and/or reverse-transcription (RT) efficacy. We discovered that the CDC-recommended h-RP primer/probe set has a faulty design, because both PCR primers are located in the same exon, which allows for unwanted PCR-amplification of background genomic DNA (gDNA). By removing RNA from nose-swab samples by an RNase treatment, we showed that the presence of gDNA in samples resulted in false-positive signals for the h-RP test control. This is rather serious, because it could lead to false-negative test outcomes, since the CDC interpretation of an absent SARS-CoV-2 rRT-PCR signal plus a positive h-RP rRT-PCR signal is interpreted as "2019-nCoV not detected", whereas a false-positive h-RP rRT-PCR signal resulting from amplification of gDNA should be interpreted as "Invalid Result" and the procedure should be repeated. In order to overhaul the faulty h-RP rRT-PCR primer/probe set with minimal modification, we designed and tested several new h-RP reverse primers. Replacement of the CDC-recommended PCR reverse primer with our selected exon-exon junction reverse primer corrected the problem of false-positive results with this important SARS-CoV-2 RT-PCR test control and thus eliminated the problem of potential false-negative COVID-19 diagnoses.

microbiology↗

bioRxiv2020

Immunoglobulin switch-like recombination regions implicated in the formation of extrachromosomal circular 45S rDNA involved in the maternal-specific translation system of zebrafish

Cellular translation is essential to all life on earth and in recent years we have reported on the discovery of a unique dual translation system in zebrafish. In this system, a maternal-type variant shows absolute expression in eggs and is progressively replaced during embryogenesis by a somatic-type variant. There are several translation system components, all with a non-coding RNA part, that show this dual characteristic: snRNA, snoRNA, rRNA, RNaseP, tRNA, and SRP-RNA. To produce sufficient ribosomes during oogenesis, zebrafish amplify their 45S locus (18S-5.8S-28S tandem repeat) by means of extrachromosomal circular DNA (eccDNA) organized in extrachromosomal rDNA circles (ERCs). Although this cellular process is discovered quite some time ago, still little is known about the mechanisms involved. Yet, because only the 45S maternal-type (45S-M) rRNA is expressed during oogenesis, the zebrafish genome provides a rare opportunity to compare an ERC 45S locus to a non-ERC 45S locus. In this study, we analyzed the genomic composition of the 45S-M and 45S-S (somatic-type) loci in combination with ultra-long read Nanopore sequencing of ERCs present in total DNA isolated from zebrafish eggs. We discovered 45S-M flanking sequences that were absent in the 45S-S locus and showed high homology to immunoglobulin (Ig) switch regions. Also, several other unique G-quadruplex DNA containing regions were found in the 45S-M locus. Some of those auxiliary regions showed different sizes in the sequenced ERCs, although within each ERC they appear to have identical sizes. These results point to a two-step system for ERC synthesis in zebrafish oogenesis: first the 45S-M repeat is excised from the chromosome into an ERC by recombination that uses the flanking Ig switch-like regions, after which the initial ECR is multiplied and extended into many ECRs with a varying number of 45S-M repeats.

developmental biology↗

bioRxiv2019

New observations on non-coding RNAs involved in the dual translation system in zebrafish development.

Cellular translation relies heavily on the involvements of several types of non-coding RNAs. In previous studies we have identified a dual translation system in zebrafish development, involving maternal-type and somatic-type rRNAs, snoRNAs, and snRNAs. In this study we focused on several remaining non-coding RNAs involved in the translation system; tRNAs, RNase P, and SRP RNA. Even though our studies have been limited in extent, for all three types of non-coding RNA we were able to identify a maternal-specific type, with substantial sequence differences as compared to the somatic-type variant. Hence, these RNA types complement the previously discovered RNA types in the unique dual translation system in zebrafish development.

developmental biology↗

bioRxiv2019

Maternal- and Somatic-type snoRNA Expression and Processing in Zebrafish Development

Small nucleolar RNAs (snoRNAs) are non-coding RNAs that play an important role in the complex maturation process of ribosomal RNAs (rRNAs). SnoRNAs are categorized in classes, with each class member having several variants present in a genome. Similar to our finding of specific rRNA expression types in zebrafish embryogenesis, we discovered preferential maternal- and somatic-expression for snoRNAs. Most snoRNAs and their variants have higher expression levels in somatic tissues than in eggs, yet we identified three snoRNAs; U3, U8 and snoZ30 of which specific variants show maternal- or somatic-type expression. For U3 and U8 we also found small-derived snoRNAs that lack their 5 rRNA recognition part and are essentially Domain II hairpin structures (U-DII). These U-DII snoRNAs from variants showed similar preferential expression, in which maternal-type variants are prominently expressed in eggs and subsequently replaced by a somatic-type variants during embryogenesis. This differential expression is related to the organization in tandem repeats (maternal type) or solitary (somatic-type) genes of the involved U snoRNA loci. The collective data showed convincingly that the preferential expression of snoRNAs is achieved by transcription regulation, as well as through RNA processing. Finally, we observed small-RNAs derived from internal transcribed spacers (ITSs) of a U3 snoRNA loci that via complementarity binding, may be involved in the biosynthesis of U3-DII snoRNAs. Altogether, the here described maternal- and somatic-type snoRNAs are the latest addition to the developing story about the dual ribosome system in zebrafish development.

developmental biology↗

bioRxiv2019

An alternative spliceosome defined by distinct snRNAs in early zebrafish embryogenesis

Splicing removes intronic RNA sequences from pre-mRNA molecules and enables, by alternative splicing, the generation of multiple unique RNA molecules from a single gene. As such, splicing is an essential part of the whole translation system of a cell. The spliceosome is a ribonucleoprotein complex in which five small nuclear RNAs (snRNAs) are involved; U1, U2, U4, U5, and U6. For each of these snRNAs there are variant gene copies present in a genome. Furthermore, in many eukaryotic species there is an alternative, minor spliceosome that can splice a small number of specific introns. As we previously discovered an embryogenesis-specific ribosomal system in zebrafish early embryogenesis based on variant rRNA and snoRNA expression, we hypothesized that there may also be an embryogenesis-specific spliceosome. An inventory of zebrafish snRNA genes revealed clustered and dispersed loci for all but U2 major snRNAs. For each minor spliceosome snRNA, just one gene locus was found. Since complete snRNA molecules are hard to sequence, we employed a combined PCR-sequencing approach to measure the individual snRNA-variant presence. Analysis of egg and male-adult samples revealed embryogenesis-specific and somatic-specific variants for each major snRNA. These variants have substantial sequence differences, yet none in their mRNA binding sites. Given that many of the sequence differences are found in loop structures indicate possible alternative protein binding. Altogether, with this study we established that the spliceosome is also an element of the embryogenesis-specific translation system in zebrafish.

developmental biology↗