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ABSTRACT: Metabarcoding 18S rRNA (Internal transcribed spacer 2, ITS2) Stenungsund coast (Sweden)

PROVIDER: PRJNA1236969 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR32732112_1.fastq.gz	Fastqsanger.gz
	SRR32732112_2.fastq.gz	Fastqsanger.gz
	SRR32732113_1.fastq.gz	Fastqsanger.gz
	SRR32732113_2.fastq.gz	Fastqsanger.gz
	SRR32732114_1.fastq.gz	Fastqsanger.gz

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Project description:DNA oligonucleotide microarrays were designed with 307 probes for 96 internal transcribed spacer (ITS1, located between 18S and 26S rRNA genes) sequences of known species and strains from the genus Pseudo-nitzschia (Bacillariophyceae). In addition, microarrays also carried 1893 probes targeting ITS1 aequences of marine Crenarchaeota and Alphaproteobacteria of SAR11 clade. In order to assign microarray profiles to Pseudo-nitzschia ribotypes and species and to 'train' the data analysis system, we grew cultures of Pseudo-nitzschia in the laboratory with identities confirmed through rDNA sequence analysis. In total, 9 cultures and 35 environmental water samples were hybridized to microarrays, in some cases, in duplicate or triplicate. Analysis of microarray data allowed us to identify and map Pseudo-nitzschia spp. in the coastal waters along Washington and Oregon coast of the Eastern Pacific Ocean, and to observe seasonal changes in diatom community composition. Total DNA was isolated from 9 Pseudo-nitzschia laboratory cultures and 35 environmental water samples collected during 7 field campaigns in 2007-2009. The environmental samples were collected at distances of 5 to 55 km from the coast, along the following transects in the Pacific Ocean covering over 300 km of the coastline: La Push (LP), Grays Harbor (GH), Columbia River (CR), and Newport Hydroline (NH). The DNA samples were subjected to PCR amplification with the primers specific for ITS1 sequences. The resultant biotin-labeled target samples were analyzed using microarray hybridization with the CombiMatrix ElectraSense 4X2K format. Out of 44 analyzed samples, 40, 2, and 2 were used for single, duplicate and triplicate hybridizations, respectively.

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Project description:The eukaryotic ribosome biogenesis is a highly orchestrated multistep process that starts at the nucleolus with the transcription of pre-rRNAs 5S and 35S. The latter comprises the mature 18S, 5.8S and 25S rRNAs separated by internal transcribed spacers (ITS1 and ITS2) and externally flanked by the 5’ETS and 3’ETS. The 35S pre-rRNA undergoes several co- and post-transcriptional processing events, which will enable the pre-60S and pre-40S particles to take independent maturation routes. Hundreds of assembly factors (AF) are required, being recruited and released hierarchically, for the proper folding of the rRNAs and correct positioning of the ribosomal proteins. One of the most intricate events that have recently been described is the removal of the ITS2-containing structure called pre-60S foot, which happens in a step-wise manner. Nop53 is an essential 60S AF that binds close to the ITS2 and plays a fundamental role in recruiting the RNA exosome for the 7S pre-rRNA processing, thereby dismantling the foot structure. Here we characterize the impact of Nop53 binding to the pre-60S on the compositional changes that happen during 60S assembly. For this purpose, preribosomes were affinity-purified with TAP-tagged 60S AFs (Nop7, Erb1, Rsa4, Arx1, Nmd3, Yvh1, and Lsg1) representative of different maturation stages both in the presence and absence of Nop53. Nop7 particles were also coimmunoprecipitated in the presence of Nop53 mutants incapable of recruiting the exosome (Nop53∆1-71, Nop53∆48-98) to compare with Nop53 depletion. The isolated preribosomes were analyzed by label-free MS/MS-based quantitative proteomics, revealing early and late-stage specific effects of Nop53 depletion.

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