Quantification of Hepatic Transcribing RNA Polymerase Molecules, Polyribonucleotide Elongation Rates and Messenger RNA Complexity in Fed and Fasted Rats

Abstract

We have investigated the mechanisms by which the rate of preribosomal RNA and premessenger RNA synthesis are repressed during starvation of rats by a quantitative study of the transcriptional events in isolated nuclei and by complexity analysis of the cytoplasmic polyadenylated RNA. The transcriptional activities of form A and B DNA-dependent RNA polymerases, which produce preribosomal and premessenger RNA respectively, were distinguished by the use of a-amanitin. Form C activity was shown to be absent from nuclei as isolated here.Polyribonucleotide elongation rates and the number of transcribing RNA polymerase molecules per diploid genome were determined by UMP/uridine analysis of the alkali-digested, nascent RNA labelled from r3H]UTP during 1-min incubations at 22 "C in the presence and absence of a-amanitin. The inhibition of form-A-mediated preribosomal RNA synthesis in response to starvation and cycloheximide treatment could be entirely accounted for by a decrease in the polyribonucleotide elongation rate. No significant change in the number of transcribing enzyme molecules (about 20 x lo3 per diploid genome) was detected. However on refeeding, the elongation rate was restored to normal and the number of transcribing form A molecules was elevated over the normal level. These results suggest that there are at least two modes of control acting on preribosomal RNA synthesis, one linked to protein synthesis and affecting the elongation rate, while a second regulates the number of form A RNA polymerase molecules transcribing the template.Form-B-mediated premessenger RNA synthesis responded to starvation by a decrease in the number of transcribing enzyme molecules from approximately 7 x lo3 to 3 x lo3 per diploid genome. This change was reversed on refeeding. Cycloheximide treatment did not affect premessenger RNA synthesis either via elongation or the number of transcribing enzyme molecules. The data indicate that premessenger RNA synthesis is controlled in this system at the level of RNA polymerase initiation by a mechanism not linked to protein synthesis.Hybridization analysis of the complexity of liver cytoplasmic polyadenylated mRNA from normal, starved and refed animals showed that three or four abundance classes were present in each case. No significant difference in mRNA populations between the dietary conditions was detected. More detailed examination of the most abundant class of mRNA confirmed this conclusion. The data show that although the number of transcribing form B molecules is reduced during starvation, the complexity and relative abundance of the mRNA synthesised does not change. The simplest explanation of these results is that, whilst the number of active genes does not change significantly during starvation, on average there are fewer form B enzyme molecules transcribing each gene in the starved animal.When rats are deprived of food there follows a progressive decrease in the RNA content of the liver [l -31. The activity of the nucleolar form A DNA-E~~~~~, RNA polymerase or nucleosidetriphos...

Massively parallel sequencing of the polyadenylated transcriptome of C. elegans

“…Yeast, with an average cell volume of 35-60 mm 3 depending on bud scar number (Johnston et al 1979), has been estimated to contain 15,000 mRNA molecules (Hereford and Rosbash 1977), whereas mammalian hepatic cells with a volume of about 30-40 times that of yeast are estimated to contain 300,000-500,000 mRNA molecules (Coupar et al 1978 . With 558 cells in the 30 3 30 3 60-mm egg, the average embryonic cell volume can be estimated to be ;60 mm 3 , yielding an estimate of about 20,000 mRNA molecules per embryonic cell.…”

“…For example, a single L2 larval stage animal has just 687 nuclei. If each cell contains ;25,000-50,000 mRNA molecules, based on interpolation from estimates for yeast and mammalian cells and supported by estimates of total RNA per isolated embryonic cell for C. elegans (see Methods for details ;Hardy 1976;Hereford and Rosbash 1977;Coupar et al 1978), an L2 animal contains 17-35 million mRNA molecules. With the Illumina 1G, as many as 30 million aligned sequence tags can be obtained from a single flow cell, thus sampling an average-sized RNA molecule reproducibly expressed in that stage once on average, although of course there will be statistical variation of the level of sampling.…”

Starvation and hypothyroidism exert an overlapping influence on rat hepatic messenger RNA activity profiles.

“…Both hypothyroidism and starvation are characterized by a diminished activity of RNA polymerase I and 11 (19,23) and reduced protein and RNA synthesis (13)(14)(15)(16)(17). These alterations may be related either as cause or effect to the changes in the mRNA activity profiles observed in our study.…”

“…Conversely, refeeding a starved animal and administration of T3 to a hypothryoid rat effectively restore these changes to normal (16,18,19). The molecular basis for the impairment and restoration has not been clearly delineated.…”

“…Moreover, both starvation and hypothyroidism reduce the functional messenger (m)RNA for ME (20)(21)(22), whereas starvation also reduces the functional mRNA for pyruvate kinase (23) as well as other selected proteins to a greater extent than the overall reduction in mRNA and total RNA content. Although the complexity of the mRNA sequences seems to be unchanged by either starvation (19) or hypothyroidism (24), no information is available regarding the influence of the profile of individual mRNA sequences as revealed by two-dimensional gel electrophoresis of in vitro translated products generated by poly(A)+ RNA from starving rats. This technique has been successfully applied in our laboratory to study the genomic changes that occur in the rat liver as a result of manipulation of the thyroidal state of the animal (25).…”