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- Dynamics and interactions of nuclear proteins revealed by quantitative photobleaching microscopyPublication . Rino, José; Fonseca, M. Carmo, 1959-; Soares, Eduardo Ducla, 1944-The nucleus is a complex cellular organelle, exhibiting a high degree of organization and also a highly dynamic nature. Live cell imaging using fluorescent proteins (FPs) as molecular tags and photobleaching techniques have been essential in revealing the dynamic nature of the cell nucleus. In this thesis, these tools were used to study molecular dynamics and interactions inside this cellular compartment. Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) were used to analyze the kinetic behavior of spliceosome components SmE, U2AF65, U2AF35, SF1 and SC35 in the nucleus of living cells. The recruitment mechanism of splicing factors (SFs) to the sites of transcription is still poorly understood. Our results rule out the hypothesis that a transcription specific signal recruits SFs from the speckles. They also suggest the formation of multi-protein complexes distinct from the spliceosome. The existence of these complexes was confirmed by Fluorescence Resonance Energy Transfer (FRET) techniques, which revealed that SFs could interact with each other even in the absence of active splicing. A novel U2AF65 self-interaction was also detected, suggesting altogether that levels of SFs in speckles are consistent with self-organization mechanisms. The intranuclear mobility of mRNPs was studied using two GFP-tagged mRNA-binding proteins, PABPN1 and TAP, as mRNA markers. A novel FLIP method was devised to quantify the mobility of the RNA-bound and unbound pools of molecules and used to test whether myosin motors were implicated in mRNP movement. We show that this is not the case and that myosin inhibition appears to affect transcription instead. A novel FLIP after Photoactivation method was developed to study the nucleocytoplasmic exchange dynamics of nuclear proteins, yielding the permanence times of molecules inside the nucleus. The method was used to study the role of the structural domains of TAP in its shuttling activity.
- Frontiers in fluorescence microscopyPublication . Rino, José; Braga, José; Henriques, Ricardo; Carmo-Fonseca, MariaHow we see organisms and cells depends on the tools at our disposal. For over 150 years, biologists were forced to rely on fixed, dehydrated and stained specimens in order to guess how the living cells could function. It all changed abruptly during the last two decades when the rapid development of novel imaging techniques revolutionized the way scientists look at the structures of life alive.
- Depletion of the yeast nuclear exosome subunit Rrp6 results in accumulation of Polyadenylated RNAs in a discrete domain within the nucleolusPublication . Carneiro, Tiago; Carvalho, Célia; Braga, José; Rino, José; Milligan, Laura; Tollervey, David; Carmo-Fonseca, MariaRecent data reveal that a substantial fraction of transcripts generated by RNA polymerases I, II, and III are rapidly degraded in the nucleus by the combined action of the exosome and a noncanonical poly(A) polymerase activity. This work identifies a domain within the yeast nucleolus that is enriched in polyadenylated RNAs in the absence of the nuclear exosome RNase Rrp6 or the exosome cofactor Mtr4. In normal yeast cells, poly(A)(+) RNA was undetectable in the nucleolus but the depletion of either Rrp6 or Mtr4 led to the accumulation of polyadenylated RNAs in a discrete subnucleolar region. This nucleolar poly(A) domain is enriched for the U14 snoRNA and the snoRNP protein Nop1 but is distinct from the nucleolar body that functions in snoRNA maturation. In strains lacking both Rrp6 and the poly(A) polymerase Trf4, the accumulation of poly(A)(+) RNA was suppressed, suggesting the involvement of the Trf4-Air1/2-Mtr4 polyadenylation (TRAMP) complex. The accumulation of polyadenylated snoRNAs in a discrete nucleolar domain may promote their recognition as substrates for the exosome.
- The intranuclear mobility of messenger RNA binding proteins is ATP dependent and temperature sensitivePublication . Calapez, Alexandre; Pereira, Henrique M.; Calado, Ângelo; Braga, José; Rino, José; Carvalho, Célia; Tavanez, João Paulo; Wahle, Elmar; Rosa, Agostinho C.; Carmo-Fonseca, MariaAfter being released from transcription sites, messenger ribonucleoprotein particles (mRNPs) must reach the nuclear pore complexes in order to be translocated to the cytoplasm. Whether the intranuclear movement of mRNPs results largely from Brownian motion or involves molecular motors remains unknown. Here we have used quantitative photobleaching techniques to monitor the intranuclear mobility of protein components of mRNPs tagged with GFP. The results show that the diffusion coefficients of the poly(A)-binding protein II (PABP2) and the export factor TAP are significantly reduced when these proteins are bound to mRNP complexes, as compared with nonbound proteins. The data further show that the mobility of wild-type PABP2 and TAP, but not of a point mutant variant of PABP2 that fails to bind to RNA, is significantly reduced when cells are ATP depleted or incubated at 22 degrees C. Energy depletion has only minor effects on the intranuclear mobility of a 2,000-kD dextran (which corresponds approximately in size to 40S mRNP particles), suggesting that the reduced mobility of PABP2 and TAP is not caused by a general alteration of the nuclear environment. Taken together, the data suggest that the mobility of mRNPs in the living cell nucleus involves a combination of passive diffusion and ATP-dependent processes.