VOLUME 29 NUMBER 10 OCTOBER 2011 nature biotechnology A r t i c l e sFluorescent proteins (FPs) 1 whose fluorescence can be reversibly or irreversibly switched by optical irradiation have opened new opportunities for the imaging of cells. They have facilitated in vivo protein-tracking schemes 2,3 , applications based on singlemolecule observations 4,5 and fluorescence microscopy with subdiffraction resolution [6][7][8][9][10] .Still, photoswitchable proteins have not displayed their full potential, because proteins that are just photoactivatable 11-13 can be switched only once, which implies that repeated measurements with the same molecule are impossible. On the other hand, photochromic or reversibly switchable fluorescent proteins (RSFPs) can be repeatedly photoswitched between a fluorescent and a nonfluorescent state by irradiation with light of two different wavelengths. However, in all previously characterized RSFPs, the wavelength used for generating the fluorescence emission is identical to one of the wavelengths used for switching the fluorescence on or off. The result is a complex interlocking of switching and fluorescence readout [14][15][16][17][18][19][20][21][22] , impeding or even precluding many applications, including fluorescence nanoscopy (super-resolution microscopy). Hence, the identification of an RSFP in which the generation of fluorescence is disentangled from switching has long been pursued. RESULTS Generation of the RSFP DreiklangNumerous GFP variants exhibit some degree of (generally undesirable) reversible photoswitching 4,23,24 . We found that the fluorescence of the yellow fluorescent protein Citrine 25,26 , a derivative of GFP, can be reversibly modulated to a small extent by alternate irradiation with light of 365 nm (on switching) and 405 nm (off switching), whereas fluorescence is excited at 515 nm. However, the achievable contrast was low, especially at pH values >6, rendering the reversible switching of Citrine unusable (Supplementary Fig. 1).To further develop this unusual switching behavior, we performed extensive random mutagenesis as well as directed PCR-mediated mutagenesis on a plasmid encoding Citrine. We transformed Escherichia coli with the plasmid, and screened with an automated home-built fluorescence microscope for bacterial colonies expressing fluorescent proteins whose fluorescence was excited with green light (515 nm) and which could be reversibly photoswitched from a fluorescent state to a long-lived nonfluorescent state by irradiation with near-UV (405 nm) light and back to a fluorescent state by UV (365 nm) light (Fig. 1a). In several consecutive screening rounds ~70,000 individual clones were analyzed. Finally, we identified a mutant differing from Citrine at four positions (Citrine-V61L, F64I, Y145H, N146D) ( Supplementary Fig. 2), which can be effectively switched and excited to fluoresce. We named this switchable fluorescent protein Dreiklang, the German word for a three-note chord in music.At thermal equilibrium, Dreiklang adopts the brightly fluorescent ...
During microRNA (miRNA) biogenesis, two endonucleolytic reactions convert stem-loop-structured precursors into mature miRNAs. These processing steps can be posttranscriptionally regulated by RNA-binding proteins (RBPs). Here, we have used a proteomics-based pull-down approach to map and characterize the interactome of a multitude of pre-miRNAs. We identify ∼180 RBPs that interact specifically with distinct pre-miRNAs. For functional validation, we combined RNAi and CRISPR/Cas-mediated knockout experiments to analyze RBP-dependent changes in miRNA levels. Indeed, a large number of the investigated candidates, including splicing factors and other mRNA processing proteins, have effects on miRNA processing. As an example, we show that TRIM71/LIN41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. We provide an extended database of RBPs that interact with pre-miRNAs in extracts of different cell types, highlighting a widespread layer of co- and posttranscriptional regulation of miRNA biogenesis.
Mammalian neurofilament triplet proteins (68 K, 160 K and 200 K) have been correlated by a biochemical, immunological and protein chemical study. The 160 K and 200 K triplet proteins are intermediate filament proteins in their own right, since they reveal the alpha‐helical coiled‐coil rod domain analyzed in detail for the 68 K protein. Triplet proteins display two distinct arrays. Their amino‐terminal region built analogously to non‐neuronal intermediate filament proteins should allow a co‐polymerization process via the interaction of coiled‐coil domains. The extra mass of all triplet proteins is allocated to carboxy‐terminally located extensions of increasing size and unique amino acid sequences. These may provide highly charged scaffolds suitable for interactions with other neuronal components. Such a domain of 68 K reveals, in sequence analysis, 47 glutamic acids within 106 residues. The epitope recognized by a monoclonal antibody reacting probably with all intermediate filament proteins has been mapped. It is located within the last 20 residues of the rods, where six distinct intermediate filament proteins point to a consensus sequence.
Abstract. The sequence of tubulin-tyrosine ligase (TTL), the enzyme catalyzing the ATP-dependent posttranslational addition of a tyrosine to the carboxyterminal end of detyrosinated ot-tubulin, has been determined. TTL from bovine and porcine brain was purified by immunoaffinity chromatography and extensively characterized by protein sequencing. Oligonucleotides derived from the protein sequence were synthesized and partial eDNA sequences were obtained using reversed transcribed brain mRNA in polymerase chain reactions. Polymerase chain reaction fragments were used to isolate a full-length eDNA clone from a randomly primed hgtl0 eDNA library obtained from embryonic porcine brain mRNA. Porcine TTL is encoded by 1,137 nucleotides corresponding to 379 amino acid residues. It has a molecular weight of 43,425 and a calculated isoelectric point of 6.51. Northern blot analysis revealed a surprisingly long mRNA (~ 6 kb in embryonic porcine brain). The protein sequence of TTL shares no extended homology with the sequences in the data banks. TTL contains a potential serine phosphorylation site for cAMP-dependent protein kinase (RKAS at positions 73 to 76). Residues 244 to 258 lie at the surface of the molecule. A rabbit antibody raised against a synthetic peptide corresponding to this sequence binds to native TTL. The same sequence contains the cleavage site for endoproteinase Glu-C (residue 248) previously shown to convert TTL into a nicked derivative in which the two fragments still form a tight complex but don't display enzymatic activity.
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