BSTRACTThe 59-to-39 mRNA degradation machinery localizes to cytoplasmic processing bodies (P-bodies), which are non-membranous structures found in all eukaryotes. Although P-body function has been intensively studied in yeast, less is known about their role in mammalian cells, such as whether P-body enzymes are actively engaged in mRNA degradation or whether P-bodies serve as mRNA storage depots, particularly during cellular stress. We examined the fate of mammalian mRNAs in P-bodies during translational stress, and show that mRNAs accumulate within Pbodies during amino acid starvation. The 59 and 39 ends of the transcripts residing in P-bodies could be identified, but poly(A) tails were not detected. Using the MS2 mRNA-tagging system for mRNA visualization in living cells, we found that a stationary mRNA population formed in P-bodies during translational stress, which cleared gradually after the stress was relieved. Dcp2-knockdown experiments showed that there is constant degradation of part of the P-body-associated mRNA population. This analysis demonstrates the dual role of P-bodies as decay sites and storage areas under regular and stress conditions.

Processing bodies (PBs) are non-membranous cytoplasmic structures found in all eukaryotes. Many of their components such as the Dcp1 and Dcp2 proteins are highly conserved. Using live-cell imaging we found that PB structures disassembled as cells prepared for cell division, and then began to reassemble during the late stages of cytokinesis. During the cell cycle and as cells passed through S phase, PB numbers increased. However, there was no memory of PB numbers between mother and daughter cells. Examination of hDcp1a and hDcp1b proteins by electrophoresis in mitotic cell extracts showed a pronounced slower migrating band, which was caused by hyper-phosphorylation of the protein. We found that hDcp1a is a phospho-protein during interphase that becomes hyper-phosphorylated in mitotic cells. Using truncations of hDcp1a we localized the region important for hyper-phosphorylation to the center of the protein. Mutational analysis demonstrated the importance of serine 315 in the hyper-phosphorylation process, while other serine residues tested had a minor affect. Live-cell imaging demonstrated that serine mutations in other regions of the protein affected the dynamics of hDcp1a association with the PB structure. Our work demonstrates the control of PB dynamics during the cell cycle via phosphorylation.

Summary The transcriptional response of β-actin to extra-cellular stimuli is a paradigm for transcription factor complex assembly and regulation. Serum induction leads to a precisely timed pulse of β-actin transcription in the cell population. Actin protein is proposed to be involved in this response, but it is not known whether cellular actin levels affect nuclear β-actin transcription. We perturbed the levels of key signaling factors and examined the effect on the induced transcriptional pulse by following endogenous β-actin alleles in single living cells. Lowering serum response factor (SRF) protein levels leads to loss of pulse integrity, whereas reducing actin protein levels reveals positive feedback regulation, resulting in elevated gene activation and a prolonged transcriptional response. Thus, transcriptional pulse fidelity requires regulated amounts of signaling proteins, and perturbations in factor levels eliminate the physiological response, resulting in either tuning down or exaggeration of the transcriptional pulse.

S-adenosylhomocysteine hydrolase (AHCY) is thought to be located at the sites of ongoing AdoMet-dependent methylation, presumably in the cell nucleus. Endogenous AHCY is located both in cytoplasm and the nucleus. Little is known regarding mechanisms that drive its subcellular distribution, and even less is known on how mutations causing AHCY deficiency affect its intracellular dynamics. Using fluorescence microscopy and GFP-tagged AHCY constructs we show significant differences in the intensity ratio between nuclei and cytoplasm for mutant proteins when compared with wild type AHCY. Interestingly, nuclear export of AHCY is not affected by leptomycin B. Systematic deletions showed that AHCY has two regions, located at both sides of the protein, that contribute to its nuclear localization, implying the interaction with various proteins. In order to evaluate protein interactions in vivo we engaged in bimolecular fluorescence complementation (BiFC) based studies. We investigated previously assumed interaction with AHCY-like-1 protein (AHCYL1), a paralog of AHCY. Indeed, significant interaction between both proteins exists. Additionally, silencing AHCYL1 leads to moderate inhibition of nuclear export of endogenous AHCY.

Examining NFAT transcription factor dynamics in single cells during signal propagation from cell membrane to the nucleus reveals unique modes of operation for protein isoforms.

The kinetic aspects of RNA polymerase II as it transcribes mRNA have been revealed over the past decade by use of live-cell imaging and kinetic analyses. It is now possible to visualize polymerase molecules in action, and most importantly to detect and follow the mRNA product as it is generated in real time on active genes. Questions such as the speed at which mRNAs are transcribed or the number of polymerases running along a particular gene can be addressed at high temporal resolution. These kinetic studies highlight the tight regulation that genes encounter when moving between active and inactive states, and ultimately will shed light on the kinetic aspects of transcription of genes under perturbed states. The scientific pathway along which these findings were unearthed begins with the imaging of the action of hundreds of genes working in concert in fixed cells. The state of the art has reached the capability of analyzing the transcription of single alleles in living mammalian cells.

No abstract

The translocation of single mRNPs (mRNA-protein complexes) from the nucleus to the cytoplasm through the nuclear pore complex (NPC) is an important basic cellular process. Originally, in order to visualize this process, single mRNP export was examined using electron microscopy (EM) in fixed Chironomus tentans specimens. These studies described the nucleocytoplasmic translocation of huge mRNPs (~30 kb) transcribed from the Balbiani-ring genes. However, knowledge of the in vivo mRNP kinetics in cell compartments remained poor up until recently. The current use of unique fluorescent protein tags, which are able to bind to mRNA transcripts, has allowed the detection and measurements of single mRNP kinetics in living cells. This has demonstrated that mRNP movement is affected by the size of the transcript and the splicing process. It was found that mRNP rates of translocation are slower in the nucleus compared to the cytoplasm and that the cell nucleus contains interchromatin tracks in which mRNPs diffuse. In order to track single mRNP movement in living cells, it is important to be able to identify single mRNP molecules transcribed from a certain gene, at the single-cell level. Single-molecule analysis of gene expression requires advanced imaging systems and analytical software in order to detect and follow the movement of single mRNPs. In this chapter we describe the methods required for the detection and tracking of single mRNP movement in living mammalian cells.