Biology subjects

de Jong, L.

Publications and source records attributed to de Jong, L..

2 recordsLinked to original sources

bioRxiv2017

Nanopore sequencing of full-length BRCA1 mRNA transcripts reveals co-occurrence of known exon skipping events

Laboratory assays evaluating the effect of DNA sequence variants on BRCA1 mRNA splicing may contribute to classification by providing molecular evidence. However, our knowledge of normal and aberrant BRCA1 splicing events to date has been limited to data derived from assays targeting partial transcript sequences. For the first time, we resolve the exon structure of whole BRCA1 transcripts using MinION nanopore sequencing of long-range PCR amplicons. Our study identified 32 BRCA1 isoforms, including 18 novel isoforms which comprised skipping of multiple contiguous and/or non-contiguous exons. Furthermore, we show that known BRCA1 exon skipping events, such as {Delta}(9,10) and {Delta}21, can co-occur in a single transcript, with some isoforms containing four or more alternative splice junctions. Our results highlight complexity in BRCA1 transcript structure that has not previously been described. This finding has key implications for predicting translation frame of splicing transcripts, important for interpreting the clinical significance of spliceogenic variants. Future research is warranted to quantitatively assess full length BRCA1 transcript levels, and to assess the application of nanopore sequencing for routine evaluation of potential spliceogenic variants.

genomics↗

bioRxiv2016

In culture cross-linking of bacterial cells reveals proteome scale dynamic protein-protein interactions at peptide level

Identification of dynamic protein-protein interactions at the peptide level on a proteomic scale is a challenging approach that is still in its infancy. We have developed a system to cross-link cells directly in culture with the special lysine cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). We used the Gram positive model bacterium Bacillus subtilis as an exemplar system. Within 5 min extensive intracellular cross-linking was detected, while intracellular cross-linking in a Gram-negative species, Escherichia coli, was still undetectable after 30 min, in agreement with the low permeability in this organism for lipophilic compounds like BAMG. We were able to identify 82 unique inter-protein cross-linked peptides with less than a 1% false discovery rate by mass spectrometry and genome-wide data base searching. Nearly 60% of the inter-protein cross-links occur in assemblies involved in transcription and translation. Several of these interactions are new, and we identified a binding site between the {delta} and {beta}' subunit of RNA polymerase close to the downstream DNA channel, providing a clue into how {delta} might regulate promoter selectivity and promote RNA polymerase recycling. Our methodology opens new avenues to investigate the functional dynamic organization of complex protein assemblies involved in bacterial growth.

biochemistry↗