RISS 검색 - 국내학술지논문

1
U4 snRNA variants of Bombyx mori

Shamayra S. SMAIL,Rene J. HERRERA 한국곤충학회 2007 Entomological Research Vol.37 No.4
It is known that the spliceosome’'s catalytic core is composed of U2, U6 and U5 small nuclear (sn)RNAs. U6 is chaperoned and held inactive by intermolecular base-pair interactions by the U4 snRNA molecule until its incorporation into the spliceosomal complex. In previous studies, a number of variant forms of U1, U2 and U6 were detected in the silk moth Bombyx mori. Considering U4’'s unique role as a modulator of U6’'s activity, the aim of the present study was to determine if the multiplicity of U6 variants observed in B. mori is complemented in U4. Five U4 variants were identified in the B. moriWhole Genome Shotgun (WGS) database and were used to design internal U4 snRNA primers. These oligonucleotides were subsequently used to create four U4 reverse transcription (RT)-polymerase chain reaction (PCR) libraries with RNA extracted from the silk gland of B. mori. Two major U4 variants were identified from these libraries. The majority of the variation among genomic and transcript-derived U4 variant sequences is located in stem-loop II and III as well as at the 3′-end. No differences between the isoforms are located in the critical U4/U6 intermolecular binding regions. Sequence comparison analyses based on U4 intraspecific and interspecific variability delineate several evolutionarily conserved regions.
2
Small nuclear RNA variants of three Bombyx mori strains

Annia MESA,Jason A. SOMARELLI,Rene J. HERRERA 한국곤충학회 2008 Entomological Research Vol.38 No.1
The spliceosome is a high molecular weight cellular complex responsible for the removal of non-protein coding introns from pre-mRNA to form mature mRNA transcripts. It comprises five major uridine (U)-rich small nuclear (sn)RNAs, to which a number of proteins bind and interact. Variant snRNAs have been identified in several organisms, although it remains to be seen whether or not these isoforms have distinct cellular roles. Nevertheless, many of these sequences have spatiotemporal trends in expression, suggesting that the variant snRNAs are not functionally equivalent. In this report, we examine and contrast the available data on snRNAs in two strains of Bombyx mori : European 703 and Nistari from India. In addition, the genomic snRNA sequences from the p50T strain are described. Thus far, isoforms of U1, U2, U4 and U6 have been characterized in B. mori European 703 and/or Nistari strains using expression libraries. In this study, an in silico approach was used to identify the genomic counterparts of the U snRNA variants in the 6X Whole Genome Shotgun (WGS) of the B. mori p50T strain. The present study is the first comparison of snRNAs in different B. mori strains. Overall, we found that 46 full length U snRNA loci and 76 suspected truncated genes are present in the B. mori genome of the p50T strain. A total of 14 full length genes match previously identified snRNAs in either the Nistari and/or the European 703 strains. Multiple sequence alignments of upstream controlling elements revealed conserved boxes in a subset of U snRNA genes. The presence of divergent promoters within specific snRNA 5′-flanking sequences suggests that these loci may be transcribed from different controlling elements or are not expressed. The number of nucleotide differences within a given type of U snRNA is strongly correlated with its copy number in the genome (r2= 77.8%) and it may reflect a relaxation of selection pressure on genes of higher copy number. The multiplicity in gene copy may provide for numerous, full length snRNA loci with variable sequences that adopt unique roles in pre-mRNA splicing, possibly by modulating protein–NA and/or RNA–NA interactions and in doing so affecting gene expression and development.
3
A three-dimensional model of the U1 small nuclear ribonucleoprotein particle

Jason A. SOMARELLI,Annia MESA,Ambrish ROY,Yang ZHANG,Rene J. HERRERA 한국곤충학회 2010 Entomological Research Vol.40 No.2
Most of the pre-mRNAs in the eukaryotic cell are comprised of protein-coding exons and non-protein-coding introns. The introns are removed and the exons are ligated together, or spliced, by a large, macromolecular complex known as the spliceosome. This RNA-protein assembly is made up of five uridine-rich small nuclear RNAs (U1-, U2-, U4-, U5- and U6-snRNA) as well over 300 proteins, which form small nuclear ribonucleoprotein particles (snRNPs). Initial recognition of the 5' exon/intron splice site is mediated by the U1 snRNP, which is composed of the U1 snRNA as well as at least ten proteins. By combining structural informatics tools with the available biochemical and crystallographic data, we attempted to simulate a complete, three dimensional U1 snRNP from the silk moth, Bombyx mori. Comparison of our model with empirically derived crystal structures and electron micrographs pinpoints both the strengths and weaknesses in the in silico determination of macromolecular complexes. One of the most striking differences between our model and experimentally generated structures is in the positioning of the U1 snRNA stem-loops. This highlights the continuing difficulties in generating reliable, complex RNA structures; however, three-dimensional modeling of individual protein subunits by threading provided models of biological significance and the use of both automated and manual docking strategies generated a complex that closely reflects the assembly found in nature. Yet, without utilizing experimentally-derived contacts to select the most likely docking scenario, ab initio docking would fall short of providing a reliable model. Our work shows that the combination of experimental data with structural informatics tools can result in generation of near-native macromolecular complexes.
4
U2 snRNA variants are differentially incorporated into spliceosomes

Jason A. SOMARELLI,Annia MESA,Shamayra S. SMAIL,Angel L. ARES,Rene J. HERRERA 한국곤충학회 2009 Entomological Research Vol.39 No.2
In this study, five U2 small nuclear (sn)RNA variants were detected in the posterior silk gland of the Bombyx mori Nistari strain, one of which represents a novel U2 isoform not previously identified in other strains of this species. Following glycerol gradient ultracentrifugation of B. mori silk gland whole cell lysate, the newly isolated variant, U2α, was detected at a greater frequency in total cell lysate than in a high density glycerol gradient fraction rich in spliceosomal complexes. Conversely, previously identified isoforms U2A, U2B, U2D and U2N are abundant in the fraction containing high molecular weight spliceosomal complexes, possibly indicating their greater involvement in splicing. As expected, western blot and semi-quantitative reverse transcription-polymerase chain reaction experiments indicate high levels of specific serine and arginine rich (SR) proteins and total U2 snRNA (all variants included) in the fraction enriched in spliceosomes. Free energy values for each U2 isoform, as well as their individual stem-loops, were estimated to determine their structural stability. Due to the essential role of U2 in the transesterification reactions, it is possible that these isoforms may modulate splicing through differential incorporation into the spliceosome.

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