Synthesis of the ribosomal RNA precursor in human cells: mechanisms, factors and regulation.

The ribosomal RNA precursor (pre-rRNA) comprises three of the four ribosomal RNAs and is synthesized by RNA polymerase (Pol) I. Here, we describe the mechanisms of Pol I transcription in human cells with a focus on recent insights gained from structure-function analyses. The comparison of Pol I-specific structural and functional features with those of other Pols and with the excessively studied yeast system distinguishes organism-specific from general traits. We explain the organization of the genomic rDNA loci in human cells, describe the Pol I transcription cycle regarding structural changes in the enzyme and the roles of human Pol I subunits, and depict human rDNA transcription factors and their function on a mechanistic level. We disentangle information gained by direct investigation from what had apparently been deduced from studies of the yeast enzymes. Finally, we provide information about how Pol I mutations may contribute to developmental diseases, and why Pol I is a target for new cancer treatment strategies, since increased rRNA synthesis was correlated with rapidly expanding cell populations.

Effects of polyploidy on the coordination of gene expression between organellar and nuclear genomes in Leucanthemum Mill. (Compositae, Anthemideae).

Whole-genome duplications (WGD) through polyploid speciation are associated with disruptions of well-tuned relationships among the three plant cell genomes. Key metabolic processes comprising multi-subunit enzyme complexes, for which partner proteins are both nuclear- and plastid-encoded, are dependent on maintenance of stoichiometric ratios among the subunits to avoid cytonuclear imbalances after WGDs. By using qPCR for gene copy and transcript number quantification, we have studied the relationship of subunit expression in the two gene pairs rbcL/rbcS (the two subunits of RuBisCO) and psbA/psbO (two members of photosystem II) in closely related members of Leucanthemum (Compositae, Anthemideae), comprising a diploid, a tetraploid, and a hexaploid species. While gene copy numbers exhibit the expected pattern of an increase in the nuclear-encoded partner gene relative to the plastid-encoded one, we find that the two partner gene systems behave differently after WGD: While in the psbA/psbO partner gene system, shifts in the gene copy balance caused by polyploidization are not accommodated for through changes in transcription intensities of the two partner genes, the rbcL/rbcS system even shows an unexpected reversed dosage effect with up-regulated transcription intensities on both the nuclear and the plastidal side. We interpret the behavior of the psbA/psbO partner gene system as being due to the stoichiometrically relaxed relationship between the two gene products caused by a fast, damage-provoked combustion of the psbA gene product (the D1 core protein of PSII). Conversely, the finely tuned expression dependencies of the rbcL/rbcS system may be the reason for the observed positive feedback runaway signal as reaction to gene copy imbalances caused by a polyploidization shock.

The Composition of the Arabidopsis RNA Polymerase II Transcript Elongation Complex Reveals the Interplay between Elongation and mRNA Processing Factors.

Transcript elongation factors (TEFs) are a heterogeneous group of proteins that control the efficiency of transcript elongation of subsets of genes by RNA polymerase II (RNAPII) in the chromatin context. Using reciprocal tagging in combination with affinity purification and mass spectrometry, we demonstrate that in Arabidopsis thaliana, the TEFs SPT4/SPT5, SPT6, FACT, PAF1-C, and TFIIS copurified with each other and with elongating RNAPII, while P-TEFb was not among the interactors. Additionally, NAP1 histone chaperones, ATP-dependent chromatin remodeling factors, and some histone-modifying enzymes including Elongator were repeatedly found associated with TEFs. Analysis of double mutant plants defective in different combinations of TEFs revealed genetic interactions between genes encoding subunits of PAF1-C, FACT, and TFIIS, resulting in synergistic/epistatic effects on plant growth/development. Analysis of subnuclear localization, gene expression, and chromatin association did not provide evidence for an involvement of the TEFs in transcription by RNAPI (or RNAPIII). Proteomics analyses also revealed multiple interactions between the transcript elongation complex and factors involved in mRNA splicing and polyadenylation, including an association of PAF1-C with the polyadenylation factor CstF. Therefore, the RNAPII transcript elongation complex represents a platform for interactions among different TEFs, as well as for coordinating ongoing transcription with mRNA processing.