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Developmental changes in the protein and ribonucleic acid components of rat brain messenger ribonucleic acid-protein particles isolated from free polyribosomes by oligo(dT)-cellulose chromatography.

ABSTRACT: A study has been made of the developmental changes that occur in the RNA and protein moieties of mRNA-protein particles isolated from newborn and adult rat forebrain free polyribosomes. mRNA-protein particles were isolated by oligo(dT)-cellulose chromatography from salt-washed polyribosomes dissociated by puromycin/0.5 M-KCl treatment as two fractions (E1 and E2) by using Tris/HCl/NaCl eluting buffers containing respectively 25 and 50% (v/v) formamide. Isopycnic centrifugation on CsCl gradients showed that the newborn-derived fractions E1 and E2 has buoyant densities of 1.48--1.50 and 1.41--1.43 g/cm3. Adult-derived E1 and E2 fractions had corresponding values of 1.47 and 1.42 g/cm3. The pooled mRNA-protein particles from the E1 and E2 fractions after deproteinization with proteinase K sedimented with a mean size of approx. 18 S on a sucrose gradient containing 85% formamide with little differences between mRNA molecules from newborn and adult. The mean lengths of the poly(A) segments were similar, being about 130 nucleotides long. Distinct changes were found in the protein composition of the mRNA-protein particles. Fractions E1 and E2 from the newborn contained two major proteins of mol.wts. 74 000 and 52 000 with differences in the relative proportions in each fraction. In contrast, adult fractions E1 and E2 contained predominantly the larger protein. However, the adult fraction E2 contained a more heterogeneous population of minor bands of proteins, including that of mol.wt. 52 000. The findings are discussed briefly in relation to other changes in the developing brain.

SUBMITTER: Elliott RM

PROVIDER: S-EPMC1162080 | biostudies-other | 1980 Jul

REPOSITORIES: biostudies-other

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Project description:Introduction: Case-control analyses have shown BARD1 variants to be associated with up to >2-fold increase in risk of breast cancer, and potentially greater risk of triple negative breast cancer. BARD1 is included in several gene sequencing panels currently marketed for the prediction of risk of cancer, however there are no gene-specific guidelines for the classification of BARD1 variants. We present the most comprehensive assessment of BARD1 messenger RNA splicing, and demonstrate the application of these data for the classification of truncating and splice site variants according to American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines. Methods: Nanopore sequencing, short-read RNA-seq (whole transcriptome and targeted), and capillary electrophoresis analysis were performed by four laboratories to investigate alternative BARD1 splicing in blood, breast, and fimbriae/ovary related specimens from non-cancer affected tissues. Splicing data were also collated from published studies of nine different tissues. The impact of the findings for PVS1 annotation was assessed for truncating and splice site variants. Results: We identified 62 naturally occurring alternative spliced BARD1 splicing events, including 19 novel events found by next generation sequencing and/or reverse transcription PCR analysis performed for this study. Quantitative analysis showed that naturally occurring splicing events causing loss of clinically relevant domains or nonsense mediated decay can constitute up to 11.9% of overlapping natural junctions, suggesting that aberrant splicing can be tolerated up to this level. Nanopore sequencing of whole BARD1 transcripts characterized 16 alternative isoforms from healthy controls, revealing that the most complex transcripts combined only two alternative splicing events. Bioinformatic analysis of ClinVar submitted variants at or near BARD1 splice sites suggest that all consensus splice site variants in BARD1 should be considered likely pathogenic, with the possible exception of variants at the donor site of exon 5. Conclusions: No BARD1 candidate rescue transcripts were identified in this study, indicating that all premature translation-termination codons variants can be annotated as PVS1. Furthermore, our analysis suggests that all donor and acceptor (IVS+/-1,2) variants can be considered PVS1 or PVS1_strong, with the exception of variants targeting the exon 5 donor site, that we recommend considering as PVS1_moderate.

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Developmental changes in the protein and ribonucleic acid components of rat brain messenger ribonucleic acid-protein particles isolated from free polyribosomes by oligo(dT)-cellulose chromatography.

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