SummaryBackgroundRegulation of cell size requires coordination of growth and proliferation. Conditional loss of cyclin-dependent kinase 1 in mice permits hepatocyte growth without cell division, allowing us to study cell size in vivo using transcriptomics and metabolomics.ResultsLarger cells displayed increased expression of cytoskeletal genes but unexpectedly repressed expression of many genes involved in mitochondrial functions. This effect appears to be cell autonomous because cultured Drosophila cells induced to increase cell size displayed a similar gene-expression pattern. Larger hepatocytes also displayed a reduction in the expression of lipogenic transcription factors, especially sterol-regulatory element binding proteins. Inhibition of mitochondrial functions and lipid biosynthesis, which is dependent on mitochondrial metabolism, increased the cell size with reciprocal effects on cell proliferation in several cell lines.ConclusionsWe uncover that large cell-size increase is accompanied by downregulation of mitochondrial gene expression, similar to that observed in diabetic individuals. Mitochondrial metabolism and lipid synthesis are used to couple cell size and cell proliferation. This regulatory mechanism may provide a possible mechanism for sensing metazoan cell size.

The relevance of serine 5 phosphorylation of RNA polymerase II carboxy-terminal domain during initiation has been difficult to determine in mammalian cells as no general in vivo Ser5 kinase has been identified. Here, we demonstrate that deletion of the TFIIH kinase subunit Mat1 in mouse fibroblasts leads to dramatically reduced Pol II Ser5 phosphorylation. This is associated with defective capping and reduced Ser2 phosphorylation, decreased Pol II progression into elongation and severely attenuated transcription detected through analysis of nascent mRNAs, establishing a general requirement for mammalian Mat1 in transcription. Surprisingly, the general defect in Pol II transcription in Mat1−/− fibroblasts is not reflected in the majority of steady-state mRNAs. This indicates widespread stabilization of mRNAs and points to the existence of a regulatory mechanism to stabilize mRNAs following transcriptional attenuation, thus revealing a potential caveat in similar studies limited to analysis of steady-state mRNAs.

Recently, it has been reported that there is a differential subcellular distribution of components of the minor U12-dependent and major U2-dependent spliceosome, and further that the minor spliceosome functions in the cytoplasm. To study the subcellular localization of the snRNA components of both the major and minor spliceosomes, we performed in situ hybridizations with mouse tissues and human cells. In both cases, all spliceosomal snRNAs were nearly exclusively detected in the nucleus, and the minor U11 and U12 snRNAs were further shown to colocalize with U4 and U2, respectively, in human cells. Additionally, we examined the distribution of several spliceosomal snRNAs and proteins in nuclear and cytoplasmic fractions isolated from human cells. These studies revealed an identical subcellular distribution of components of both the U12-and U2-dependent spliceosomes. Thus, our data, combined with several earlier publications, establish that, like the major spliceosome, components of the U12-dependent spliceosome are localized predominantly in the nucleus.localization ͉ pre-mRNA splicing ͉ snRNA ͉ U12-dependent spliceosome P re-mRNA splicing is an essential step in the co/posttranscriptional processing of eukaryotic transcripts before their export from the nucleus. The low-abundance U12-dependent ''minor'' spliceosome exists in parallel with the U2-dependent ''major'' spliceosome in most multicellular eukaryotes (1). It catalyzes the removal of a rare class of introns (U12-type) that represent Ͻ1% of introns in mammals (2, 3). The U12-dependent spliceosome contains four unique snRNAs: U11, U12, U4atac, and U6atac, which are paralogs of U1, U2, U4, and U6 snRNAs of the U2-dependent spliceosome, respectively (4, 5). U5 snRNA, in contrast, is shared between the two spliceosomes. In addition to the snRNAs, both spliceosomes contain numerous snRNP and non-snRNP proteins (6, 7).The five snRNP components of the major spliceosome are predominantly localized in the nucleus, but the biogenesis of four of them involves a cytoplasmic step (8). That is, after transcription by RNA Pol II, the snRNAs U1, U2, U4, and U5, which all contain an Sm protein-binding site, are first exported into the cytoplasm. The Sm proteins subsequently bind, and the snRNA's cap is hypermethylated to 2,2,7-tri-methyl-guanosine (m 3 G) (9, 10). These processing steps are a prerequisite for their reimport into the nucleus (11). In contrast to these so-called Sm-class snRNAs, U6 snRNA is transcribed by RNA pol III, acquires Sm-like proteins (Lsm2-8) (12) and a ␥-monomethyl phosphate cap, and does not leave the nucleus. The minor snRNAs U11, U12, and U4atac are also Sm-class snRNAs, containing an Sm-binding site that is also bound by Sm proteins and an m 3 G cap (5, 13), whereas U6atac is transcribed by RNA pol III, possesses a ␥-monomethyl phosphate cap (5), and is assembled with Lsm proteins (14). After nuclear import, spliceosomal snRNPs first pass through Cajal bodies, where they undergo further modifications and assembly (15, 16) and then subsequentl...

The rate of excision of U12-type introns has been reported to be slower than that of U2-type introns, suggesting a rate-limiting bottleneck that could down-regulate genes containing U12-type introns. The mechanistic reasons for this slower rate of intron excision are not known, but lower abundance of the U12-type snRNPs and slower rate of assembly or catalytic activity have been suggested. To investigate snRNP abundance we concentrated on the U4atac snRNA, which is the least abundant of the U12-type snRNAs and is limiting the formation of U4atac/U6atac complex. We identified mouse NIH-3T3 cell line isolates in which the level of both U4atac snRNA and U4atac/U6atac complexes is reduced to 10%-20% of the normal level. We used these cell lines to investigate splicing efficiency by transient transfection of a reporter gene containing a U12-type intron and by quantitative PCR analysis of endogenous genes. The splicing of the reporter U12-type intron was very inefficient, but the activity could be restored by overexpression of U4atac snRNA. Using these U4atac-deficient NIH-3T3 cells, we confirmed the results of previous studies showing that U12-type introns of endogenous genes are, indeed, excised more slowly than U2-type introns, but we found that the rate did not differ from that measured in cells displaying normal levels of U4atac snRNA. Thus our results suggest that the cellular abundance of the snRNPs does not limit U12-type intron splicing under normal conditions.

BackgroundThe U12-type spliceosome is responsible for the removal of a subset of introns from eukaryotic mRNAs. U12-type introns are spliced less efficiently than normal U2-type introns, which suggests a rate-limiting role in gene expression. The Drosophila genome contains about 20 U12-type introns, many of them in essential genes, and the U12-type spliceosome has previously been shown to be essential in the fly.Methodology/Principal FindingsWe have used a Drosophila line with a P-element insertion in U6atac snRNA, an essential component of the U12-type spliceosome, to investigate the impact of U12-type introns on gene expression at the organismal level during fly development. This line exhibits progressive accumulation of unspliced U12-type introns during larval development and the death of larvae at the third instar stage. Surprisingly, microarray and RT-PCR analyses revealed that most genes containing U12-type introns showed only mild perturbations in the splicing of U12-type introns. In contrast, we detected widespread downstream effects on genes that do not contain U12-type introns, with genes related to various metabolic pathways constituting the largest group.Conclusions/SignificanceU12-type intron-containing genes exhibited variable gene-specific responses to the splicing defect, with some genes showing up- or downregulation, while most did not change significantly. The observed residual U12-type splicing activity could be explained with the mutant U6atac allele having a low level of catalytic activity. Detailed analysis of all genes suggested that a defect in the splicing of the U12-type intron of the mitochondrial prohibitin gene may be the primary cause of the various downstream effects detected in the microarray analysis.