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A Myosin I Isoform in the Nucleus

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Science  13 Oct 2000:
Vol. 290, Issue 5490, pp. 337-341
DOI: 10.1126/science.290.5490.337

Abstract

A nuclear isoform of myosin I β that contains a unique 16–amino acid amino-terminal extension has been identified. An affinity-purified antibody to the 16–amino acid peptide demonstrated nuclear staining. Confocal and electron microscopy revealed that nuclear myosin I β colocalized with RNA polymerase II in an α-amanitin– and actinomycin D–sensitive manner. The antibody coimmunoprecipitated RNA polymerase II and blocked in vitro RNA synthesis. This isoform of myosin I β appears to be in a complex with RNA polymerase II and may affect transcription.

Myosin I is a single-headed, nonfilamentous member of the myosin superfamily of actin-based molecular motors (1, 2). There are at least four different subclasses of myosin I proteins, all containing a 110- to 150-kD heavy chain and one to six light chains. Myosin I is diffusely distributed throughout the cytoplasm (3). It concentrates near cortical surfaces and in the perinuclear region (3), and it appears to mediate plasma membrane extension (3, 4), vesicle and organelle transport (5), and mechanochemical regulation of calcium channels in hair cells (6).

Affinity-purified polyclonal antibodies to bovine adrenal myosin I recognized a 120-kD protein that is larger than the antigen (116 kD) (7). Confocal and electron microscopy showed cytoplasmic and nuclear staining with these antibodies. Biochemical assays on nuclei demonstrated that the 120-kD protein binds adenosine triphosphate (ATP) and calmodulin, is associated with K+-EDTA ATPase activity, and binds actin only in the absence of ATP (7). Because these characteristics are defining features of the myosin superfamily of proteins (1), we considered the possibility that the 120-kD protein is a myosin I isoform that is a nuclear molecular motor.

The 120-kD protein was immunoprecipitated from nuclei isolated from mouse fibroblasts (8). SDS–polyacrylamide gel electrophoresis (PAGE) showed a 120-kD band by Coomassie blue staining in the immunoprecipitates. Microsequencing of the 120-kD protein (8) revealed high sequence homology (>98%) with myosin I β. It also revealed the presence of 12 amino acids preceding the consensus initiator methionine (9–11) of myosin I β.

Analysis of an embryonic mouse cDNA library with mouse myosin I β primers (12) yielded two sequences. The shorter of the two sequences encoded a peptide that contained the consensus mouse myosin I β start site (9, 10). The longer sequence contained another upstream start site that is followed by nucleotides that encode a 16–amino acid sequence (Fig. 1A) that is not found in other myosin I β proteins (1–6, 9–11). Moreover, 12 of the 16 amino acids exactly matched the 12 amino acids that were found by microsequencing the 120-kD protein. Therefore, the 120-kD protein (119,720 daltons, as calculated from the amino acid sequence) is referred to as nuclear myosin I β (NMIβ) to differentiate it from the smaller (117,999 daltons), exclusively cytoplasmic myosin I β isoform (CMIβ).

Figure 1

Structure of the myosin I β gene (29). (A) The ATG's corresponding to NMIβ and consensus start sites are underlined. The peptide obtained by microsequencing that overlaps the consensus start site is shown in bold. The known mouse myosin I β cDNA and protein sequences (9, 10) are in italics. PCR amplification and DNA sequencing of the NMIβ cDNA showed that the coding region is identical to other mouse myosin I β coding sequences (9, 10) and nearly identical (>98% homology) to the rat myosin I β coding sequence (11). (B) The structure of the myosin I β gene in the mouse, rat, and human. The myosin I β gene is in an 80-kb region of chromosome 11 (13) that may contain a mutation that causes a serious neurological and behavioral disorder in the vibrator mouse (9). At least two mRNA species that code for myosin I β in vibrator and normal mice, with identical protein coding regions but different 5′UTRs, have also been reported (9, 10). NMIβ starts with the ATG in exon –1. The NMIβ cDNA is similar to a cDNA (myr 2) that contains two potential start sites and codes for a single, cytoplasmic protein in adult rats (11). The GenBank accession number for the nucleotide sequence is AY007255.

The mouse myosin I β gene is located on chromosome 11 (9,13). It contains 13 exons, starting with exon 1, and is ∼12 kb long. Two additional exons (exons −2 and −1) were located upstream of exon 1 on chromosome 11 (Fig. 1B). Exon −2 is found in the 5′ untranslated region (5′UTR) of the normal adult mouse myosin I β cDNA (10). Exon –1 is not found in other mouse myosin I β cDNAs (9, 10). Exon −1 contains a second translation start site that is in frame with the consensus myosin I β translation start site on exon 1. Translation starting from the ATG on exon −1, which is preceded by a Kozak sequence (14), produces a protein identical to mouse myosin I β but with 16 additional amino acids at the NH2-terminus. The first six amino acids come from exon −1 and the remaining 10 amino acids come from exon 1. Exon −1 also contributes 24 nucleotides, very rich in GC, that constitute the 5′UTR of NMIβ. Thus, the NMIβ and CMIβ isoforms appear to be translated from separate transcripts originating from the same gene on chromosome 11.

To determine whether the 16–amino acid extension directs NMIβ to the nucleus, we cloned the cDNA for the NMIβ and CMIβ isoforms into a vector containing the FLAG epitope and transfected the result into NIH 3T3 cells (15). Confocal microscopy showed that CMIβ-FLAG was confined to the cytoplasm (Fig. 2, A to D). In contrast, there was nuclear and cytoplasmic expression of the NMIβ-FLAG (Fig. 2, E to H). Experiments were also performed with an affinity-purified antibody to the 16–amino acid peptide (16). This antibody recognized a 120-kD protein by protein immunoblotting (Fig. 3) and also stained the nucleus (Fig. 2, I to L). There was no staining when the affinity-purified antibody to NMIβ peptide was adsorbed with the peptide before staining (Fig. 2, M to P). These data suggest that the unique, 16–amino acid NH2-terminal extension directs NMIβ to the nucleus.

Figure 2

Confocal images of NIH 3T3 cells. Top row: cells expressing CMIβ-FLAG; second row: cells expressing NMIβ-FLAG; third row: cells stained with affinity-purified antibody to NMIβ peptide; bottom row, cells stained with the same antibody preadsorbed with peptide. The same cell was photographed in each row. (A, E, I, and M) Differential interference contrast images. (B, F,J, and N) Cells stained with DAPI to visualize the nuclei. (C, G, K, andO) FLAG localization. (D, H,L, and P) Merged DAPI/NMIβ images. The purple color indicates the colocalization of DAPI and NMIβ. There is no nuclear staining in cells expressing CMIβ-FLAG (top row), whereas the cells expressing NMIβ-FLAG show cytoplasmic and nuclear staining (second row). The NMIβ cDNA contains start sites for NMIβ and CMIβ, and translation of the two FLAG-tagged proteins results in staining of the nucleus and the cytoplasm. The affinity-purified antibody to NMIβ peptide stains mainly the nucleus (third row); there is no staining when the antibody is preadsorbed with the peptide (bottom row).

Figure 3

Antibody to NMIβ peptide recognizes a 120-kD protein. A protein immunoblot analysis with this antibody (16) was performed on extracts prepared from nuclei isolated from HeLa cells (HeLa NE), NIH 3T3 cells (3T3), and HeLa cells (HeLa). The migration of marker proteins (MWM) and their molecular weight (in kilodaltons) are given. The lower band (∼60 kD) is probably a degradation product of the 120-kD band.

Confocal microscopy (17) also showed that NMIβ and RNA polymerase II (Pol II) colocalize in control cells (Fig. 4A). This colocalization was lost (Fig. 4B) when cells were treated with α-amanitin, a transcription inhibitor that stimulates the degradation of the large subunit of RNA Pol II (18), and decreased when cells were treated with actinomycin D (Fig. 4C), which blocks transcription by binding to DNA (19). Because transcriptional foci are 70 to 100 nm long (20), NMIβ and RNA Pol II should be within 100 nm if they are functionally related. Immunoelectron microscopy (21) demonstrated statistically significant numbers of NMIβ and RNA Pol II within 100 nm of each other. They were >100 nm apart in cells treated with α-amanitin or actinomycin D (Fig. 4, D to F, and Table 1).

Figure 4

Immunolocalization of NMIβ and RNA Pol II in HeLa cells. (A to C) Localization of RNA Pol II (red) and NMIβ (green) by confocal microscopy in control cells (A) and cells treated with α-amanitin (B) or with actinomycin D (C). The yellow color indicates the colocalization of RNA Pol II and NMIβ in control cells. This colocalization is practically lost (B) or reduced (C) when transcription is inhibited by α-amanitin or actinomycin D, respectively. Scale bar, 1 μm. (D to F) Double immunogold labeling of RNA Pol II (5-nm particles) and NMIβ (10-nm particles) in untreated cells (D) and cells treated with α-amanitin (E) or actinomycin D (F). Clusters of RNA Pol II are frequently intermingled with clusters of NMIβ (arrows) in control cells (D). The density of RNA Pol II labeling (arrowheads) is reduced and the colocalization is insignificant following the disruption of the Pol II complex by α-amanitin (E). The density of RNA Pol II labeling is similar to that in the control sections (arrowheads) but colocalization with NMIβ is rare following treatment with actinomycin D (F). Control samples incubated as above but minus the primary antibodies showed no significant gold labeling. Scale bar, 100 nm.

Table 1

A statistical analysis performed on three independent double-labeling immunoelectron microscopy colocalization experiments (Fig. 4) (21) demonstrated statistically significant numbers of NMIβ and RNA Pol II within 100 nm of each other in control cells. This distribution was disrupted and the colocalization of NMIβ and RNA Pol II was insignificant at <100 nm after inhibition of RNA transcription. NS, not significant (P > 0.05).

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To further investigate the interaction between NMIβ and RNA Pol II, we used antibody to NMIβ peptide to immunoprecipitate NMIβ from nuclei. Protein immunoblot analysis (22) showed that the large subunit of RNA Pol II coprecipitated with NMIβ (Fig. 5A). RNA Pol II was not present in immunocomplexes when the antibody was preincubated with the peptide (Fig. 5A). In addition, antibody to NMIβ peptide inhibited RNA synthesis in vitro (Fig. 5B) (23). In contrast, neither the same antibody preincubated with peptide nor affinity-purified antibody to myosin II inhibited transcription (Fig. 5B).

Figure 5

Association of NMIβ and RNA Pol II. (A) Coimmunoprecipitation of NMIβ and RNA Pol II. NMIβ was immunoprecipitated from nuclei (8) using antibody to NMIβ peptide (Pep Ab) or the same antibody preadsorbed with the peptide (Ads Pep Ab) (22). Protein immunoblot analysis was conducted with a mAb to the large subunit of RNA Pol II (top) or with antibody to NMIβ peptide (bottom). NMIβ and RNA Pol II coimmunoprecipitated when antibody to NMIβ peptide was used, but neither was present when the immunoprecipitation was performed using preadsorbed antibody. The migration of RNA Pol II and NMIβ in a nuclear extract (NE) is shown for comparison. Molecular weight markers (MWM, in kilodaltons) are shown in the middle. (B) Inhibition of in vitro transcription by antibody to NMIβ peptide. HeLa cell nuclear extracts were preincubated with buffer (Con) or various agents and the transcription products analyzed (23). There is no transcription product in the presence of α-amanitin (Am) or antibody to NMIβ peptide. In contrast, preadsorbing this antibody with the peptide eliminated the inhibition (Ads Pep Ab), and an affinity-purified antibody to myosin II (Myo II Ab) had no effect on transcription. This experiment was repeated three to seven times; numbers at the bottom of each lane give the fractional activity relative to control, as judged by PhosphorImager quantification. The arrowhead marks the top of the gel; the arrow marks the transcription products.

Nuclear processes such as transcription involve large molecular complexes and require energy (24). The abundance of actin in the nucleus (25) suggests a role for myosin in the nucleus. Myosin I may be well suited as a nuclear, actin-dependent molecular motor because it does not have to form filaments to be biochemically or biologically active (1). Myosin I molecules contain a positively charged domain in the tail region that interacts with acidic phospholipids and powers the movement of membrane fragments on actin filaments (1, 5). Similarly, NMIβ could bind to actin through the actin binding site on its head and negatively charged nuclear components through its tail region. RNA polymerases have been suggested to power the movement of DNA and transcription complexes relative to each other (26–28) in a manner analogous to energy conversion by myosin. Our data suggest that nuclear myosin I, perhaps together with RNA polymerases, could potentially power transcription.

  • * These authors contributed equally to this paper.

  • To whom correspondence should be addressed. E-mail: primal{at}uic.edu

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