Uridine-rich small nuclear ribonucleoproteins (U snRNPs) are splicing factors, which are diffusely distributed in the nucleoplasm and also concentrated in nuclear speckles. Fluorescently labeled, native U1 snRNPs were microinjected into the cytoplasm of living HeLa cells. After nuclear import single U1 snRNPs could be visualized and tracked at a spatial precision of 30 nm at a frame rate of 200 Hz employing a custom-built microscope with single-molecule sensitivity. The single-particle tracks revealed that most U1 snRNPs were bound to specific intranuclear sites, many of those presumably representing pre-mRNA splicing sites. The dissociation kinetics from these sites showed a multiexponential decay behavior on time scales ranging from milliseconds to seconds, reflecting the involvement of U1 snRNPs in numerous distinct interactions. The average dwell times for U1 snRNPs bound at sites within the nucleoplasm did not differ significantly from those in speckles, indicating that similar processes occur in both compartments. Mobile U1 snRNPs moved with diffusion constants in the range from 0.5 to 8 m 2 /s. These values were consistent with uncomplexed U1 snRNPs diffusing at a viscosity of 5 cPoise and U1 snRNPs moving in a largely restricted manner, and U1 snRNPs contained in large supramolecular assemblies such as spliceosomes or supraspliceosomes. INTRODUCTIONCell nuclei exhibit a high degree of spatial and functional organization of their molecular components (Cremer and Cremer, 2001;Misteli, 2005). The question how nuclear factors move and interact within this well-organized structure, how molecular factors find their targets, and how trafficking exerts a possible regulatory function is currently a focus in cell biology (Bubulya and Spector, 2004;Pederson, 2004;Gorski and Misteli, 2005). An exact quantification of interactions and molecular mobilities is the prerequisite for a detailed understanding of the formation of intranuclear structures and the regulation of their function by modifications of interactions and transport (Gorski et al., 2006).Eukaryotic pre-mRNA transcripts go through several post-transcriptional modifications before their translocation by the NPCs into the cytoplasm (Darzacq et al., 2005). Usually pre-mRNAs have noncoding sequences designated as introns that must be removed from the sequence to yield functional mRNA. This essential biochemical processing is designated as pre-mRNA splicing, which is achieved by intranuclear molecular pre-assembled complexes, the socalled spliceosomes. Spliceosomes consist of more than 70 different proteins, many of which are part of the uridine-rich small nuclear ribonucleoproteins (U snRNPs), which are classified as U1, U2, U5, and U4/U6, according to their small nuclear RNA (snRNA) content. With the exception of U6, the snRNAs are synthesized in the nucleus by RNA polymerase II and exported to the cytoplasm, where sets of common and specific proteins bind to the snRNAs (Will and Luhrmann, 2001). After their cytoplasmic assembly U snRNPs are reimported into ...
Fibril-forming collagen proteins of the types I, III, and V were extracted from fetal calf skin, purified by differential salt precipitation, and analyzed by infrared matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (IR-MALDI-TOF-MS). Glycerol was used as liquid IR-MALDI matrix. Noncovalently bound triple helices of the types I and V were detected from the NaCl precipitate. After heating at 43 degrees C for 10 min, resulting in the dissociation of the triple helix, the single alpha-chain subunits were detected. For type I, mass spectra acquired from molecular sieve chromatography fractions revealed the presence of further substructures of dimeric type and of supramolecular complexes up to the tetramer. Triple helices of type III, stabilized by covalent disulfide bonds, were detected from the total protein precipitate also after heat treatment. For type III, even hexamer and nonamer structures with molecular weights close to 600 and 900 kDa were recorded. For comparison, ultraviolet (UV-)MALDI-MS measurements with 2,5-dihydroxybenzoic acid as matrix were carried out with some of the samples. Here, only the single alpha-chains were detected with significantly reduced sensitivity.
Several DNA/RNA sequencing strategies have been developed using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). In the reverse Sanger sequencing approach alpha-thiophosphate-containing NTPs are employed. Sequencing ladders are produced by the subsequent exonuclease cleavage, which is inhibited by the alpha-S-NTP at the 3' terminus. Here the reverse Sanger sequencing of RNA is described. The stability of RNA during the UV-MALDI process is higher relative to DNA, and RNA can be easily synthesized by transcription using bacteriophage RNA polymerase. alpha-S-rNTP was added to the reaction in a ratio of 1:3 to the native rNTPs and was incorporated statistically by the RNA polymerase. Four separate sequence ladders were produced, to avoid the problem of the only 1u mass difference between uridine and cytidine. However, it was shown that RNA transcription does not produce homogeneous transcripts. Therefore isolation of the full-length transcript is required to attain a non-ambiguous interpretation of cleavage spectra. This is achieved by the exclusive immobilization of the full-length transcript on a solid phase. The full-length transcripts were hybridized to magnetic beads, coated with short universal sequences, complementary to the in vitro RNA. After purification and isolation the RNA full-length transcript is cleaved by snake venom phosphodiesterase (SVP) and the obtained sequence ladder is analyzed by MALDI-MS.
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