Kinesin transports RNA: isolation and characterization of an RNA-transporting granule

RNA transport is an important and fundamental event for local protein synthesis, especially in neurons. RNA is transported as large granules, but little is known about them. Here, we isolated a large RNase-sensitive granule (size: 1000S approximately) as a binding partner of conventional kinesin (KIF5). We identified a total of 42 proteins with mRNAs for CaMKIIalpha and Arc in the granule. Seventeen of the proteins (hnRNP-U, Pur alpha and beta, PSF, DDX1, DDX3, SYNCRIP, TLS, NonO, HSPC117, ALY, CGI-99, staufen, three FMRPs, and EF-1alpha) were extensively investigated, including their classification, binding combinations, and necessity for the "transport" of RNA. These proteins and the mRNAs were colocalized to the kinesin-associated granules in dendrites. The granules moved bidirectionally, and the distally directed movement was enhanced by the overexpression of KIF5 and reduced by its functional blockage. Thus, kinesin transports RNA via this granule in dendrites coordinately with opposite motors, such as dynein.     

Comparative analysis of two C-terminal kinesin motor proteins: KIFC1 and KIFC5A

We have taken advantage of the close structural relationship between two C-terminal motors, KIFC5A and KIFC1, to examine the sequence requirements for targeting of these two motors within the cell. Although KIFC5A and KIFC1 are almost identical in their motor and stalk domains, they differ in well-defined regions of their tail domains. Specific antisera to these motors were used to determine their localization to distinct subcellular compartments, the spindle for KIFC5A or membranous organelles for KIFC1. In addition to defining the intracellular localization of KIFC1, the reactivity of the KIFC1 antibody demonstrates that this motor contains a frame shift with respect to KIFC5A and is likely the product of a separate gene. The divergent tail domains of these motors are predicted to harbor specific information that directs them to their correct intracellular targets. In order to define the sequences responsible for the differential localization of these two motors, GFP was fused to motors with various tail deletions and their localization visualized after transfection. We were able to identify distinct sequences in each motor responsible for its unique cellular localization. The KIFC5A tail contains a 43 amino acid sequence with both nuclear localization and microtubule binding activity while KIFC1 contains a 19 amino acid sequence sufficient to target this motor to membrane-bounded organelles.     

PLP-1 binds nematode double-stranded telomeric DNA

The integrity and proper functioning of telomeres require association of telomeric DNA sequences with specific binding proteins. We have characterized PLP-1, a PURa homolog encoded by F45E4.2, which we previously identified as a candidate double stranded telomere binding protein, by affinity chromatography followed by mass spectrometry. PLP-1 bound double-stranded telomeric DNA in vitro as shown by competition assays. Core binding was provided by the third and fourth nucleotides of the TTAGGC telomeric repeat. This is quite different from the binding sequence of CEH-37, another C. elegans telomere binding protein, suggesting that multiple proteins may bind nematode telomeric DNA simultaneously in vivo.     

Kinesin superfamily protein 3 (KIF3) motor transports fodrin-associating vesicles important for neurite building

Kinesin superfamily proteins (KIFs) comprise several dozen molecular motor proteins. The KIF3 heterotrimer complex is one of the most abundantly and ubiquitously expressed KIFs in mammalian cells. To unveil the functions of KIF3, microinjection of function-blocking monovalent antibodies against KIF3 into cultured superior cervical ganglion (SCG) neurons was carried out. They significantly blocked fast axonal transport and brought about inhibition of neurite extension. A yeast two-hybrid binding assay revealed the association of fodrin with the KIF3 motor through KAP3. This was further confirmed by using vesicles collected from large bundles of axons (cauda equina), from which membranous vesicles could be prepared in pure preparations. Both immunoprecipitation and immunoelectron microscopy indicated the colocalization of fodrin and KIF3 on the same vesicles, the results reinforcing the evidence that the cargo of the KIF3 motor consists of fodrin-associating vesicles. In addition, pulse-labeling study implied partial comigration of both molecules as fast flow components. Taken together, the KIF3 motor is engaged in fast axonal transport that conveys membranous components important for neurite extension.     

S1 nuclease sensitivity of a double-stranded telomeric DNA sequence.

We examined structural properties of poly d(C4A2).d(T2G4), the telomeric DNA sequence of the ciliated protozoan Tetrahymena. Under conditions of high negative supercoiling, poly d(C4A2).d(T2G4) inserted in a circular plasmid vector was preferentially sensitive to digestion with S1 nuclease. Only the C4A2 strand was sensitive to first-strand S1 cutting, with a markedly skewed pattern of hypersensitive sites in tracts of either 46 or 7 tandem repeats. Linear poly d(C4A2).(T2G4) showed no preferential S1 sensitivity, no circular dichroism spectra indicative of a Z-DNA conformation, no unusual Tm, and no unusual migration in polyacrylamide gel electrophoresis. The S1 nuclease sensitivity properties are consistent with a model proposed previously for supercoiled poly d(CT).d(AG) (Pulleyblank et al., Cell 42:271-280, 1985), consisting of a double-stranded, protonated, right-handed underwound helix. We propose that this structure is shared by related telomeric sequences and may play a role in their biological recognition.     

A structural basis for S1 nuclease sensitivity of double-stranded DNA

A protonated form of a cloned simple repeating DNA sequence d(TC)n X d(GA) is detectable in equilibrium with the usual Watson-Crick base-paired form at pHs up to 7. This form is anomalously sensitive to a variety of single-strand-specific endonucleases. The observed pH dependent protection of N-7 of dG residues within the insert suggests that these residues are either Hoogsteen or reverse Hoogsteen base-paired to protonated dC residues of the polypyrimidine strand. A structure in which dA:dT Watson-Crick base pairs alternate with Hoogsteen syndG:dCH+ pairs appears to be the most stereochemically acceptable structure consistent with the chemical properties of this protonated DNA. Protonated d(TC)n X d(GA)n interacts with an anti-Z DNA antibody raised against brominated d(GC)n X d(GC)n.     

Bleomycin-specific fragmentation of double-stranded DNA.

Brief exposure of covalently closed circular duplex PM2 DNA to low concentrations of the clinical bleomycin mixture (Blenoxane) resulted in specific fragmentation of the genome that does not depend on the presence of superhelical turns. The double-strand breaks are in fact produced at several discrete sites on the PM2 genome but frequently occurring near the HpaII restriction endonuclease cleavage site. Initial rates of formation of nicked circular and linear duplex PM2 DNAs are reduced to different extents as the ionic strength of the reaction is increased. Increasing ionic strength is most effective in reducing the initial rate and overall yield of apparent double-strand scissions compared with single-strand scissions in the bleomycin-treated PM2 DNA.     

Method enabling pyrosequencing on double-stranded DNA

Pyrosequencing is a new nonelectrophoretic, single-tube DNA sequencing method that takes advantage of co-operativity between four enzymes to monitor DNA synthesis (M. Ronaghi, M. Uhlén, and P. Nyrén, Science 281, 363-365). Pyrosequencing has so far only been performed on single-stranded DNA. In this paper different enzymatic strategies for template preparation enabling pyrosequencing on double-stranded DNA were studied. High quality data were obtained with several different enzyme combinations: (i) shrimp alkaline phosphatase and exonuclease I, (ii) calf intestine alkaline phosphatase and exonuclease I, (iii) apyrase and inorganic pyrophosphatase together with exonuclease I, and (iv) apyrase and ATP sulfurylase together with exonuclease I. In many cases, when the polymerase chain reaction was efficient exonuclease I could be omitted. In certain cases, additives such as dimethyl sulfoxide, single-stranded DNA-binding protein, and Klenow DNA polymerase improved the sequence quality. Apyrase was the fastest and most efficient of the three different nucleotide degrading enzymes tested. The data quality obtained on double-stranded DNA was comparable with that on single-stranded DNA. Pyrosequencing data for more than 30 bases could be generated on both long and short templates, as well as on templates with high GC content.     

The kinetics of YOYO-1 intercalation into single molecules of double-stranded DNA

Recent studies have suggested that treatment of glucocorticoid to immature growth hormone (GH)-producing cell line, MtT/S cells, dramatically induced the accumulation of GH-containing secretory granules in the cytosol and differentiated into mature GH-producing cells. However, the molecular mechanism of glucocorticoid-induced GH-containing secretory granule biogenesis in the MtT/S cells remains unknown. In the present study, we found that GH mRNA expression was facilitated by application of glucocorticoid. We artificially increased GH synthesis by transfection of green fluorescent protein-tagged GH (GH-GFP) gene. We found that the artificial elevation of GH expression in the cells did not accumulate the secretory granules in the cytosol, whereas glucocorticoid-induced the biogenesis of granules in GH-GFP-expressing MtT/S cells. We next performed DNA microarray and real-time RT-PCR analysis and found that glucocorticoid significantly altered the expression of membrane trafficking-related protein, syntaxin11 (Syx11). Immunocytochemical analysis further demonstrated that Syx11 positive structures were well colocalized with GH-containing granules in both MtT/S cells and rat anterior pituitary gland. Our findings indicate that glucocorticoid regulate the expression of Syx11 and facilitate the biogenesis and the trafficking of GH-containing granules in the MtT/S cells.     

1H NMR studies of selective interactions of norfloxacin with double-stranded DNA

The interaction of the antibiotic drug norfloxacin with double-stranded DNA containing interior 5'-CpG-3', 5'-GpC-3', and 5'-GpG-3' steps was studied by 1H NMR. The drug is in fast exchange on the NMR timescale. A highly selective broadening of the imino proton resonances assigned to central CpG steps was observed after addition of drug, indicating an intercalation-like interaction. DNA sequences with central CpG steps also displayed broadening of non-hydrogen-bonded cytosine amino protons in the major groove upon addition of norfloxacin. Furthermore, a sequence-independent selective broadening of the adenine H2 resonance and an upfield shift of the guanine amino proton resonance, both protons located in the minor groove, was observed. Two-dimensional-NOESY spectra showed that no significant structural changes were induced in the DNA by the drug. The results suggest that the planar two-ring system of norfloxacin partially intercalates into CpG steps and that the drug also exhibits non-specific groove binding.     

Spontaneous sharp bending of double-stranded DNA

Sharply bent DNA is essential for gene regulation in prokaryotes and is a major feature of eukaryotic nucleosomes and viruses. The explanation normally given for these phenomena is that specific proteins sharply bend DNA by application of large forces, while the DNA follows despite its intrinsic inflexibility. Here we show that DNAs that are 94 bp in length-comparable to sharply looped DNAs in vivo-spontaneously bend into circles. Proteins can enhance the stability of such loops, but the loops occur spontaneously even in naked DNA. Random DNA sequences cyclize 10(2)-10(4) times more easily than predicted from current theories of DNA bending, while DNA sequences that position nucleosomes cyclize up to 10(5) times more easily. These unexpected results establish DNA as an active participant in the formation of looped regulatory complexes in vivo, and they point to a need for new theories of DNA bending.     

ATP-independent cooperative binding of yeast Isw1a to bare and nucleosomal DNA

Among chromatin remodeling factors, the ISWI family displays a nucleosome-enhanced ATPase activity coupled to DNA translocation. While these enzymes are known to bind to DNA, their activity has not been fully characterized. Here we use TEM imaging and single molecule manipulation to investigate the interaction between DNA and yeast Isw1a. We show that Isw1a displays a highly cooperative ATP-independent binding to and bridging between DNA segments. Under appropriate tension, rare single nucleation events can sometimes be observed and loop DNA with a regular step. These nucleation events are often followed by binding of successive complexes bridging between nearby DNA segments in a zipper-like fashion, as confirmed by TEM observations. On nucleosomal substrates, we show that the specific ATP-dependent remodeling activity occurs in the context of cooperative Isw1a complexes bridging extranucleosomal DNA. Our results are interpreted in the context of the recently published partial structure of Isw1a and support its acting as a "protein ruler" (with possibly more than one tick).     

Recognition and cleavage of double-stranded DNA by yeast VMA1-derived endonuclease.

DNA endonuclease derived from the yeast VMA1-gene product recognizes and cleaves 31 base-pairs of double-stranded DNA (dsDNA). Mixtures of the endonuclease (VDE) with a full DNA substrate consisting of 34 base-pairs, with nicked substrates each having a nick in either DNA chain, and with cleaved substrates each having a cleaved-off chain are prepared. Molecular weights (MWs) of eluted peaks from gel filtration columns were estimated from elution profiles in the presence of Mg2+ ions. Each mixture exhibited an elute peak at about 63k MW, larger than the MW of VDE unbound to dsDNA. This indicates that VDE and dsDNA substrates form stable complexes. The mixture of VDE either with the full substrate or with the nicked substrate having a nick in the anti-sense chain eluted an additional 25k-MW peak, which presumably corresponds to a cleaved product. The complex of VDE with the full substrate was eluted at 62k-MW location in the absence of Mg2+ ions and yielded a single crystal. Stable complexes of VDE either with the dsDNA substrates or with the cleaved products are obtainable.     

MDR1 P-glycoprotein transports endogenous opioid peptides

MDR1 P-glycoprotein is generally regarded as an efflux pump for amphipathic toxic compounds. The question remains, however, whether certain endogenous compounds are also substrates for this transporter. Certain peptides have been shown to interact with MDR1 Pgp as well and we have therefore investigated whether endogenous bioactive peptides are substrates. We demonstrate here that the synthetic μ-opioid peptide DAMGO is a good substrate for MDR1 Pgp. In view of its low interaction with the membrane it is an attractive ligand for measurement of MDR1 Pgp-mediated transport activity in membrane vesicles. Various linear peptides with amidated C-termini were found to inhibit MDR1 Pgp-mediated DAMGO transport. This group includes endogenous opioid peptides such as adrenorphin and endomorphin 1 and 2, as well as the neurokinin, Substance P. The latter bioactive peptides have a relatively high affinity for the transporter. Transport of endomorphin 1 and 2 could be directly demonstrated by the uptake of the radiolabeled opioid peptides in membrane vesicles from MDR1-transfected cells with a K_m of 15 and 12 μM, respectively. This opens the possibility that MDR1 Pgp is involved in the elimination and/or tissue distribution of these bioactive peptides.