A Kinetic Framework for a Mammalian RNA Polymerase in Vivo

Dundr, Miroslav; Hoffmann-Rohrer, Urs; Hu, Qiyue; Grummt, Ingrid; Rothblum, Lawrence I.; Phair, Robert D.; Misteli, Tom

doi:10.1126/science.1076164

Cited by 399 publications

(392 citation statements)

References 17 publications

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“…This reactive patch specifies the maximal rotational shift of the binding sites of two particles at close distance, at which a productive hit can occur (Vijayakumar et al, 1998). As discussed above, the reactive patch value of 3°used for the simulations presented yielded association rates that match well with known association rates among protein binding partners as well as productive hit rates for RNA polymerase I interactions with its transcriptional target, rDNA (Schreiber and Fersht, 1996;Dundr et al, 2002). However, it is possible that the actual reactive patches on snRNPs are smaller or larger than those estimated here.…”

Section: Resultssupporting

confidence: 48%

Enhancement of U4/U6 Small Nuclear Ribonucleoprotein Particle Association in Cajal Bodies Predicted by Mathematical Modeling

Klingauf

Staněk

Neugebauer

2006

MBoC

100

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Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) undergo specific assembly steps in Cajal bodies (CBs), nonmembrane-bound compartments within cell nuclei. An example is the U4/U6 di-snRNP, assembled from U4 and U6 monomers. These snRNPs can also assemble in the nucleoplasm when cells lack CBs. Here, we address the hypothesis that snRNP concentration in CBs facilitates assembly, by comparing the predicted rates of U4 and U6 snRNP association in nuclei with and without CBs. This was accomplished by a random walk-and-capture simulation applied to a three-dimensional model of the HeLa cell nucleus, derived from measurements of living cells. Results of the simulations indicated that snRNP capture is optimal when nuclei contain three to four CBs. Interestingly, this is the observed number of CBs in most cells. Microinjection experiments showed that U4 snRNA targeting to CBs was U6 snRNP independent and that snRNA concentration in CBs is ϳ20-fold higher than in nucleoplasm. Finally, combination of the simulation with calculated association rates predicted that the presence of CBs enhances U4 and U6 snRNP association by up to 11-fold, largely owing to this concentration difference. This provides a chemical foundation for the proposal that these and other cellular compartments promote molecular interactions, by increasing the local concentration of individual components.

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Section: Resultssupporting

confidence: 48%

Enhancement of U4/U6 Small Nuclear Ribonucleoprotein Particle Association in Cajal Bodies Predicted by Mathematical Modeling

Klingauf

Staněk

Neugebauer

2006

MBoC

100

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“…27 For FRAP, 1-2 pulses at full laser power were applied to a bleaching ROI encompassing the entire WPB, and the WPB FI followed to monitor recovery. For whole-cell inverse FRAP (iFRAP 28 ) a PL APO 63 ϫ 0.6-1.4NA objective was used to image at least 2 fluorescent cells at a time. All but a small region of 1 cell (containing a few WPBs) was bleached using full laser power pulses at 1-to 2-second intervals over an ϳ 5-minute period.…”

Section: Whole-wpb Fluorescence Recovery Analysis Of Rabs and Effectomentioning

confidence: 99%

The interplay between the Rab27A effectors Slp4-a and MyRIP controls hormone-evoked Weibel-Palade body exocytosis

Bierings

Hellen

Kiskin

et al. 2012

Blood

166

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Weibel-Palade body (WPB) exocytosis underlies hormone-evoked VWF secretion from endothelial cells (ECs). We identify new endogenous components of the WPB: Rab3B, Rab3D, and the Rab27A/ Rab3 effector Slp4-a (granuphilin), and determine their role in WPB exocytosis. We show that Rab3B, Rab3D, and Rab27A contribute to Slp4-a localization to WPBs. siRNA knockdown of Slp4-a, MyRIP, Rab3B, Rab3D, Rab27A, or Rab3B/ Rab27A, or overexpression of EGFPSlp4-a or EGFP-MyRIP showed that Slp4-a is a positive and MyRIP a negative regulator of WPB exocytosis and that Rab27A alone mediates these effects. We found that ECs maintain a constant amount of cellular Rab27A irrespective of the WPB pool size and that Rab27A (and Rab3s) cycle between WPBs and a cytosolic pool. The dynamic redistribution of Rab proteins markedly decreased the Rab27A concentration on individual WPBs with increasing WPB number per cell. Despite this, the probability of WPB release was independent of WPB pool size showing that WPB exocytosis is not determined simply by the absolute amount of Rab27A and its effectors on WPBs. Instead, we propose that the probability of release is determined by the fractional occupancy of WPB-Rab27A by Slp4-a and MyRIP, with the balance favoring exocytosis. (Blood. 2012;120(13):2757-2767) IntroductionHormone-evoked VWF secretion from endothelial cells (ECs) is mediated by exocytosis of specialized secretory granules (SGs) called Weibel-Palade bodies (WPBs). 1 WPB exocytosis is triggered by increases in intracellular free Ca 2ϩ or cAMP concentrations, and involves a number of molecular components, including the Nethylmaleimide-sensitive factor, VAMP3, SNAP23, syntaxin 4, RalA, the annexin A2/S100A10 complex, and phospholipase D. [2][3][4][5][6][7] In addition, Rab proteins also regulate WPB exocytosis. A subset of Rab proteins, including Rab3A-3D, Rab27A/B, and Rab37, is associated with SGs in different cell types where they regulate SG biogenesis, trafficking, and exocytosis. 8 Secretory cells often express a mixture of these "secretory" Rabs, which may have overlapping or distinct functions. Human ECs are reported to express mRNA for Rab3A, Rab3D, and Rab37,3,9,10 Rab3B protein, 11 and Rab27A mRNA and protein. 12,13 To date, Rab27A is the only endogenous EC Rab protein that has been detected on WPBs. Through its effector MyRIP and Myosin Va, Rab27A is proposed to negatively regulate WPB exocytosis. 13,14 Rab27A can interact with different effector molecules, and many secretory cells express a mixture of these effectors. 8 In these cases, SG exocytosis probably depends on the balance of Rab27A interactions with the complement of Rab effectors in the cell.In addition to MyRIP, ECs contain mRNA for the Rab27A effector Slp4-a (granuphilin). 13 Slp4-a links SGs to the plasma membrane (PM) through SG-associated Rab proteins (principally Rab27A), PM-associated syntaxins (1a, 2, or 3) and soluble Munc18 isoforms. [15][16][17][18][19] Syntaxins exist in open and closed conformations that determine their participation in SNARE complex f...

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“…All iFRAP data were corrected and normalized using the equation described in Dundr et al (2002) (also see Supplemental Material). iFRAP curves were modeled equally well with a two-phase exponential decay equation (Supplemental Figure 1).…”

Section: Inverse Fluorescence Recovery After Photobleaching Analysis mentioning

confidence: 99%

Mutant Huntingtin Impairs Post-Golgi Trafficking to Lysosomes by Delocalizing Optineurin/Rab8 Complex from the Golgi Apparatus

Toro

Alberch

Lázaro‐Diéguez

et al. 2009

MBoC

153

117

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Huntingtin regulates post-Golgi trafficking of secreted proteins. Here, we studied the mechanism by which mutant huntingtin impairs this process. Colocalization studies and Western blot analysis of isolated Golgi membranes showed a reduction of huntingtin in the Golgi apparatus of cells expressing mutant huntingtin. These findings correlated with a decrease in the levels of optineurin and Rab8 in the Golgi apparatus that can be reverted by overexpression of full-length wild-type huntingtin. In addition, immunoprecipitation studies showed reduced interaction between mutant huntingtin and optineurin/Rab8. Cells expressing mutant huntingtin produced both an accumulation of clathrin adaptor complex 1 at the Golgi and an increase of clathrin-coated vesicles in the vicinity of Golgi cisternae as revealed by electron microscopy. Furthermore, inverse fluorescence recovery after photobleaching analysis for lysosomal-associated membrane protein-1 and mannose-6-phosphate receptor showed that the optineurin/Rab8-dependent post-Golgi trafficking to lysosomes was impaired in cells expressing mutant huntingtin or reducing huntingtin levels by small interfering RNA. Accordingly, these cells showed a lower content of cathepsin D in lysosomes, which led to an overall reduction of lysosomal activity. Together, our results indicate that mutant huntingtin perturbs post-Golgi trafficking to lysosomal compartments by delocalizing the optineurin/Rab8 complex, which, in turn, affects the lysosomal function.

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A Kinetic Framework for a Mammalian RNA Polymerase in Vivo

Cited by 399 publications

References 17 publications

Enhancement of U4/U6 Small Nuclear Ribonucleoprotein Particle Association in Cajal Bodies Predicted by Mathematical Modeling

Enhancement of U4/U6 Small Nuclear Ribonucleoprotein Particle Association in Cajal Bodies Predicted by Mathematical Modeling

The interplay between the Rab27A effectors Slp4-a and MyRIP controls hormone-evoked Weibel-Palade body exocytosis

Mutant Huntingtin Impairs Post-Golgi Trafficking to Lysosomes by Delocalizing Optineurin/Rab8 Complex from the Golgi Apparatus

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