Multiparameter RNA and codon optimization: a standardized tool to assess and enhance autologous mammalian gene expression.

Autologous expression of recombinant human proteins in human cells for biomedical research and product development is often hampered by low expression yields limiting subsequent structural and functional analyses. Following RNA and codon optimization, 50 candidate genes representing five classes of human proteins--transcription factors, ribosomal and polymerase subunits, protein kinases, membrane proteins and immunomodulators--all showed reliable, and 86% even elevated expression. Analysis of three representative examples showed no detrimental effect on protein solubility while unaltered functionality was demonstrated for JNK1, JNK3 and CDC2 using optimized constructs. Molecular analysis of a sequence-optimized transgene revealed positive effects at transcriptional, translational, and mRNA stability levels. Since improved expression was consistent in HEK293T, CHO and insect cells, it was not restricted to distinct mammalian cell systems. Additionally, optimized genes represent powerful tools in functional genomics, as demonstrated by the successful rescue of an siRNA-mediated knockdown using a sequence-optimized counterpart. This is the first large-scale study addressing the influence of multiparameter optimization on autologous human protein expression.

TOR-dependent reduction in the expression level of Rrn3p lowers the activity of the yeast RNA Pol I machinery, but does not account for the strong inhibition of rRNA production.

Ribosome biogenesis is tightly linked to cellular growth. A crucial step in the regulation of ribosomal RNA (rRNA) gene transcription is the formation of the complex between RNA polymerase I (Pol I) and the Pol I-dependent transcription factor Rrn3p. We found that TOR inactivation leads to proteasome-dependent degradation of Rrn3p and a strong reduction in initiation competent Pol I-Rrn3p complexes affecting yeast rRNA gene transcription. Using a mutant expressing non-degradable Rrn3p or a strain in which defined endogenous Rrn3p levels can be adjusted by the Tet-off system, we can demonstrate that Rrn3p levels influence the number of Pol I-Rrn3p complexes and consequently rRNA gene transcription. However, our analysis reveals that the dramatic reduction of rRNA synthesis in the immediate cellular response to impaired TOR signalling cannot be explained by the simple down-regulation of Rrn3p and Pol I-Rrn3p levels.

Structure and organization of coat proteins in the COPII cage.

COPII-coated vesicles export newly synthesized proteins from the endoplasmic reticulum. The COPII coat consists of the Sec23/24-Sar1 complex that selects cargo and the Sec13/31 assembly unit that can polymerize into an octahedral cage and deform the membrane into a bud. Crystallographic analysis of the assembly unit reveals a 28 nm long rod comprising a central alpha-solenoid dimer capped by two beta-propeller domains at each end. We construct a molecular model of the COPII cage by fitting Sec13/31 crystal structures into a recently determined electron microscopy density map. The vertex geometry involves four copies of the Sec31 beta-propeller that converge through their axial ends; there is no interdigitation of assembly units of the kind seen in clathrin cages. We also propose that the assembly unit has a central hinge-an arrangement of interlocked alpha-solenoids-about which it can bend to adapt to cages of variable curvature.

Dephosphorylation of RNA polymerase I by Fcp1p is required for efficient rRNA synthesis.

Differently phosphorylated forms of RNA polymerase (Pol) II are required to guide the enzyme through the transcription cycle. Here, we show that a phosphorylation/dephosphorylation cycle is also important for RNA polymerase I-dependent synthesis of rRNA precursors. A key component of the Pol II transcription system is Fcp1p, a phosphatase that dephosphorylates the C-terminal domain of the largest Pol II subunit. Fcp1p stimulates transcription elongation and is required for Pol II recycling after transcription termination. We found that Fcp1p is also part of the RNA Pol I transcription apparatus. Fcp1p is required for efficient rDNA transcription in vivo, and also, recombinant Fcp1p stimulates rRNA synthesis both in promoter-dependent and in nonspecific transcription assays in vitro. We demonstrate that Fcp1 activity is not involved in the formation of the initiation-active form of Pol I (the Pol I-Rrn3p complex) and propose that dephosphorylation of Pol I by Fcp1p facilitates chain elongation during rRNA synthesis.

The composition of the RNA polymerase I transcription machinery switches from initiation to elongation mode.

The amounts of RNA polymerase I (Pol I) and basal rDNA transcription factors were determined in yeast whole cell extracts. A 17-fold excess of Pol I was found compared to the Pol I-specific initiation factors upstream activating factor (UAF) and core factor (CF) which underlines that both initiation factors interact with a minor fraction of Pol I when rDNA transcription is active. Surprisingly, Rrn3p, another Pol I-specific initiation factor, is more abundant in cell lysates than UAF and CF. Our analyses revealed that a large fraction of cellular Rrn3p is not associated with Pol I. However, the amount of initiation-active Rrn3p which forms a stable complex with Pol I corresponds to the levels of UAF and CF which have been shown to bind the promoter. Initiation-active Rrn3p dissociates from the template during or immediately after Pol I has switched from initiation to elongation. Our data support a model in which the elongating Pol I leaves the initiation factors UAF, CF and Rrn3p close by the promoter.