Nt-GFP-PK was produced by ligation of NM1 N-terminal sequence (aa 1C16) in front of EGFP in GFP-PK vector

Nt-GFP-PK was produced by ligation of NM1 N-terminal sequence (aa 1C16) in front of EGFP in GFP-PK vector. thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we recognized importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin around the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1. Conclusions/Significance We have shown that this novel specific NLS brings to the cell nucleus not only the nuclear isoform of myosin I (NM1 protein) but also its cytoplasmic isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus. Introduction Nuclear myosin I (NM1) was the first unconventional myosin motor detected in the cell nucleus [1]. NM1 is an isoform of earlier recognized cytoplasmic myosin Ic (Myo1c) made up of additional 16 amino acids at the N-terminus. The mRNA of NM1 is usually differently spliced yielding 5 introduction of exon made up of alternative start of translation [2]. Importantly, the ubiquitous expression and nuclear localization of NM1 in mouse organs along with high degree of conservation of the N-terminal sequence across species has been confirmed [3], [4]. This corresponds to its important functions. In the nucleus, there is ample evidence for functional involvement of NM1 in transcription by RNA polymerase I and II (Pol I and Pol II). NM1 co-localizes with both polymerases at the sites of transcription [2], [5] and actually associates with both Pol I and Pol II complexes [6], [7]. In-vivo rate of transcription is usually negatively affected by NM1 overexpression, and inhibited by NM1 knock-down and nuclear microinjections of anti-NM1 antibodies [7]. In an in-vitro transcription system, anti-NM1 antibodies inhibit transcription by both polymerases in a dose-dependent manner, whereas adding purified NM1 increases transcription [2], [6], [8]. Transcription initiation assays have revealed that NM1 exerts its function in early actions of Pol I and II transcription after the formation of pre-initiation complexes [6], [7]. Indeed, NM1 interacts with Pol I transcription factor TIF-IA, which is present only in initiation-competent portion of Pol I complexes [9], and actin that is associated with RNA polymerase I independently of active transcription [7]. According to Grummt [10], the binding of NM1 to Pol I via actin may help to initiate transcription by recruiting TIF-IA to pre-initiation complex. This model is usually further supported by the fact that functional iCRT 14 motor domain name is needed for conversation of NM1 and Pol I [11]. In addition to transcription initiation, NM1 is also involved in Pol I transcription elongation since it associates with the chromatin remodeling complex WSTF-SNF2h and might therefore recruit this complex to the actively transcribing genes [12]. Interestingly, nascent ribosomal particles seem to be accompanied by NM1 during transport from nucleolus toward nuclear pores [13] and blocking of NM1 or actin by antibodies results in nuclear retention of small ribosomal subunits [14], [15]. A role of acto-myosin Rabbit polyclonal to UBE2V2 motor in repositioning of chromosomes is usually emerging iCRT 14 [16], [17]. In pioneering work, Chuang and co-workers [18] showed that labeled artificial gene loci move, upon activation, toward the center of nucleus and that overexpression of mutated NM1 that lacks motor activity inhibits this effect. However, the exact mechanism behind these translocation phenomena is not clear. Using specific antibodies generated against its N-terminal epitope, NM1 can be detected iCRT 14 predominantly in the nucleus, nucleolus and at iCRT 14 the plasma membrane of interphase cells [1], [5], [19]. NM1 is usually a short-tailed class I myosin that binds directly to actin via its head domain name and the headgroups of acidic phospholipids via putative PH domain name within positively charged tail [20]. Neck domain name, located between head and tail, contains three IQ motifs that bind calmodulin [1]. To date, you will find no data about biochemical characteristics of this protein. Because NM1 is almost identical to Myo1c, one can expect that its basic function is usually to maintain tensions as proposed for Myo1c [21] however, the exact function of the N-terminal extension in NM1 molecule that makes the only known difference form Myo1c is usually uncertain. The observation that NM1 is usually localized mainly in the nucleus and Myo1c at the plasma membrane has.