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Relative distribution of post-nuclear poly(A)-containing RNA abundance groups within the nuclear and post-nuclear polyadenylated and non-polyadenylated RNA populations of the lactating guinea-pig mammary gland.

ABSTRACT: 1. RNA isolated from the post-nuclear supernatant of the lactating guinea-pig mammary gland was fractionated with oligo(dT)-cellulose into three populations; those that bound at ;low salt' [long poly(A) tracts, 78-32 nucleotides]; those that bound at ;high salt' [shorter poly(A) tracts, 48-21 nucleotides]; and those that did not bind [no poly(A) or short poly(A) tracts, <20 nucleotides]. Nuclear RNA was fractionated into two populations, those that bound in ;low salt' and those that did not bind. All the post-nuclear RNA fractions directed the synthesis of milk proteins in a Krebs II ascites cell-free system. 2. (3)H-labelled DNA complementary to the post-nuclear poly-(A)-containing RNA population (low-salt fraction) was fractionated into abundant (milk-protein mRNA), moderately abundant and scarce sequences. This complementary DNA was then used to investigate the distribution of the mRNA sequences in the different RNA populations. This showed that all sequences were present in polyadenylated and non-polyadenylated fractions, but that major quantitative differences were apparent. The abundant milk-protein mRNA sequences predominated in the ;low-salt' post-nuclear poly(A)-containing RNA fraction, whereas the moderately abundant sequences predominated in the non-polyadenylated post-nuclear RNA fraction. In total cellular RNA, those sequences deemed initially to be moderately abundant within the ;low-salt' poly(A)-containing RNA population were present at a concentration very similar to those of the abundant milk-protein mRNA (approx. 6x10(5) copies of each sequence/cell). Similarly, analysis of the nuclear RNA populations showed that the ;abundant' and so-called ;moderately abundant' sequences were present in essentially identical concentrations (2x10(3) copies of each sequence/cell). The majority of these (90-95%) were non-polyadenylated. 3. The results are discussed in terms of the post-transcriptional mechanisms involved in the regulation of gene expression in the lactating guinea-pig mammary gland.

SUBMITTER: Bathurst IC

PROVIDER: S-EPMC1162363 | biostudies-other | 1980 Nov

REPOSITORIES: biostudies-other

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Project description:The biological importance of changes in RNA expression is reflected by the wide variety of tools available to characterise these changes from RNA-seq data. Several tools exist for detecting differential transcript isoform usage (DTU) from aligned or assembled RNA-seq data, but few exist for DTU detection from alignment-free RNA-seq quantifications. We present the RATs, an R package that identifies DTU transcriptome-wide directly from transcript abundance estimates. RATs is unique in applying bootstrapping to estimate the reliability of detected DTU events and shows good performance at all replication levels (median false positive fraction < 0.05). We compare RATs to two existing DTU tools, DRIM-Seq & SUPPA2, using two publicly available simulated RNA-seq datasets and a published human RNA-seq dataset, in which 248 genes have been previously identified as displaying significant DTU. RATs with default threshold values on the simulated Human data has a sensitivity of 0.55, a Matthews correlation coefficient of 0.71 and a false discovery rate (FDR) of 0.04, outperforming both other tools. Applying the same thresholds for SUPPA2 results in a higher sensitivity (0.61) but poorer FDR performance (0.33). RATs and DRIM-seq use different methods for measuring DTU effect-sizes complicating the comparison of results between these tools, however, for a likelihood-ratio threshold of 30, DRIM-Seq has similar FDR performance to RATs (0.06), but worse sensitivity (0.47). These differences persist for the simulated drosophila dataset. On the published human RNA-seq dataset the greatest agreement between the tools tested is 53%, observed between RATs and SUPPA2. The bootstrapping quality filter in RATs is responsible for removing the majority of DTU events called by SUPPA2 that are not reported by RATs. All methods, including the previously published qRT-PCR of three of the 248 detected DTU events, were found to be sensitive to annotation differences between Ensembl v60 and v87.

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Relative distribution of post-nuclear poly(A)-containing RNA abundance groups within the nuclear and post-nuclear polyadenylated and non-polyadenylated RNA populations of the lactating guinea-pig mammary gland.

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