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Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a trans-acting RNA-binding protein that mediates trafficking of RNAs containing the cis-acting A2 response element (A2RE). Previous work has shown that A2RE RNAs are transported to myelin in oligodendrocytes and to dendrites in neurons. hnRNP E1 is an RNA-binding protein that regulates translation of specific mRNAs. Here, we show by yeast two-hybrid analysis, in vivo and in vitro coimmunoprecipitation, in vitro cross-linking, and fluorescence correlation spectroscopy that hnRNP E1 binds to hnRNP A2 and is recruited to A2RE RNA in an hnRNP A2-dependent manner. hnRNP E1 is colocalized with hnRNP A2 and A2RE mRNA in granules in dendrites of oligodendrocytes. Overexpression of hnRNP E1 or microinjection of exogenous hnRNP E1 in neural cells inhibits translation of A2RE mRNA, but not of non-A2RE RNA. Excess hnRNP E1 added to an in vitro translation system reduces translation efficiency of A2RE mRNA, but not of nonA2RE RNA, in an hnRNP A2-dependent manner. These results are consistent with a model where hnRNP E1 recruited to A2RE RNA granules by binding to hnRNP A2 inhibits translation of A2RE RNA during granule transport.

The interaction of E. coli's integration Host Factor (IHF) with fragments of lambda DNA containing the cos site has been studied by gel-mobility retardation and electron microscopy. The cos fragment used in the mobility assays is 398 bp and spans a region from 48,298 to 194 on the lambda chromosome. Several different complexes of IHF with this fragment can be distinguished by their differential mobility on polyacrylamide gels. Relative band intensities indicate that the formation of a complex between IHF and this DNA fragment has an equilibrium binding constant of the same magnitude as DNA fragments containing lambda's attP site. Gel-mobility retardation and electron microscopy have been employed to show that IHF sharply bends DNA near cos and to map the bending site. The protein-induced bend is near an intrinsic bend due to DNA sequence. The position of the bend suggests that IHF's role in lambda DNA packaging may be the enhancement of terminase binding/cos cutting by manipulating DNA structure.

In neural cells, such as oligodendrocytes and neurons, transport of certain RNAs along microtubules is mediated by the cis-acting heterogeneous nuclear ribonucleoprotein A2 response element (A2RE) trafficking element and the cognate trans-acting heterogeneous nuclear ribonucleoprotein (hnRNP) A2 trafficking factor. Using a yeast two-hybrid screen, we have identified a microtubule-associated protein, tumor overexpressed gene (TOG)2, as an hnRNP A2 binding partner. The C-terminal third of TOG2 is sufficient for hnRNP A2 binding. TOG2, the large protein isoform of TOG, is the only isoform detected in oligodendrocytes in culture. TOG coimmunoprecipitates with hnRNP A2 present in the cytoskeleton (CSK) fraction of neural cells, and both coprecipitate with microtubule stabilized pellets. Staining with anti-TOG reveals puncta that are localized in proximity to microtubules, often at the plus ends. TOG is colocalized with hnRNP A2 and A2RE-mRNA in trafficking granules that remain associated with CSK-insoluble tissue. These data suggest that TOG mediates the association of hnRNP A2-positive granules with microtubules during transport and/or localization. INTRODUCTIONThe heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is implicated in RNA processing, RNA transport, and translation regulation (Dreyfuss et al., 1993;Siomi and Dreyfuss, 1995;Hamilton et al., 1999;Kwon et al., 1999). HnRNP A2 binds to a specific cis-acting element, hnRNP A2 response element (A2RE), found in several dendritically localized mRNAs Munro et al., 1999). This interaction is necessary for transport of A2RE-mRNAs to the periphery of neural cells (Munro et al., 1999;Shan et al., 2003).A2RE-mRNAs, hnRNP A2, and other components form complexes that look by light microscopy like granules that are transported to the cell periphery by a microtubule-and kinesin-based mechanism. In cultured oligodendrocytes and neurons, granules containing A2RE-mRNAs and hnRNP A2 are associated with the cytoskeleton (CSK) . HnRNP A2 and some A2RE-mRNAs copurify with the CSK from rat brain (Hoek et al., 1998;Boccaccio et al., 1999).To identify molecular partners of hnRNP A2, we performed yeast two-hybrid analysis. One of the components identified is the tumor overexpressed gene (TOG) protein, a microtubule-associated protein (MAP) ubiquitously expressed in human tissues. TOG is a single copy gene that generates two mRNA splice variants (Nagase et al., 1995;Charrasse et al., 1998) encoding proteins that differ by a 60-aa insert near the C terminus. In dividing cells, TOG localizes with centrosome and spindle microtubules and may be necessary for microtubule rearrangements and spindle assembly (Charrasse et al., 1998;Lee et al., 2001). Orthologues of colonic and hepatic tumor overexpressed gene (ch-TOG) (Homo sapiens), Dis1p, Stu2p, XMAP215, ZYG-9, and mini spindles, in fission yeast, budding yeast, Xenopus laevis, Caenorhabditis elegans, and Drosophila melanogaster, respectively, have similar functions. TOG and its orthologues all contain multiple HEAT repeats that may serv...

SummaryIn oligodendrocytes and neurons genetic information is transmitted from nucleus to dendrites in the form of RNA granules. Here we describe how transport of multiple different RNA molecules in individual granules is analogous to the process of multiplexing in telecommunications. In both cases multiple messages are combined into a composite signal for transmission on a single carrier. Multiplexing provides a mechanism to coordinate local expression of ensembles of genes in myelin in oligodendrocytes and at synapses in neurons.

SummaryThe 136 codon (408 bp) denA gene encoding endonuclease II (EndoII) of bacteriophage T4 was unambiguously identi®ed through sequencing and subsequent cloning. EndoII prepared from cloned DNA through coupled in vitro transcription±translation nicked and cut DNA in vitro in a sequence-speci®c manner. In vitro (and in vivo ), the bottom strand was nicked between the ®rst and second base pair to the right of a topstrand CCGC motif shared by favoured in vitro and in vivo cleavage sites; top-strand cleavage positions varied. To the right of the cleavage position, favoured in vitro sites lacked a sequence element conserved at favoured in vivo sites. In pBR322 DNA, the sites cleaved in vivo as previously described were also cleaved in vitro, but in vitro additional sites were nicked or cleaved and the preference for individual sites was different. Also, different from the in vivo reaction, nicking was more frequent than ds cutting; in many copies of a ds cleavage site, only the bottom strand was nicked in vitro. A model is discussed in which sequential nicking of the two strands, and different factors in¯u-encing bottom-strand nicking and top-strand nicking, can explain the differences between the in vitro and the in vivo reaction.

Synthetic sites inserted into a plasmid were used to analyze the sequence requirements for in vivo DNA cleavage dependent on bacteriophage T4 endonuclease II. A 16-bp variable sequence surrounding the cleavage site was sufficient for cleavage, although context both within and around this sequence influenced cleavage efficiency. The most efficiently cleaved sites matched the sequence CGRCCGCNTTGGCNGC, in which the strongly conserved bases to the left were essential for cleavage. The less-conserved bases in the center and in the right half determined cleavage efficiency in a manner not directly correlated with the apparent base preference at each position; a sequence carrying, in each of the 16 positions, the base most preferred in natural sites in pBR322 was cleaved infrequently. This, along with the effects of substitutions at one or two of the lessconserved positions, suggests that several combinations of bases can fulfill the requirements for recognition of the right part of this sequence. The replacements that improve cleavage frequency are predicted to influence helical twist and roll, suggesting that recognition of sequence-dependent DNA structure and recognition of specific bases are both important. Upon introduction of a synthetic site, cleavage at natural sites within 800 to 1,500 bp from the synthetic site was significantly reduced. This suggests that the enzyme may engage more DNA than its cleavage site and cleaves the best site within this region. Cleavage frequency at sites which do not conform closely to the consensus is, therefore, highly context dependent. Models and possible biological implications of these findings are discussed.Sequence-specific endonucleases are crucial for the proper maintenance of DNA in cells; they are required for its replication, recombination, and repair; its defense against invading parasites; and its proper degradation during programmed cell death in differentiated organisms. Scrutiny reveals strong similarities in their mechanisms of action, suggesting that some may participate in more than one such event.The restriction system encoded by coliphage T4 and dependent on its endonuclease II (EndoII) resembles type II restriction endonucleases more than type I or III or the methylationdependent endonucleases. Restriction enzymes recognize their specific DNA targets by a combination of direct and indirect readout (30, 31), i.e., interactions between nucleotide bases and amino acid residues and interactions between amino acids and the sugar-phosphate backbone, respectively. Both interactions are dependent on the sequence of the recognition site. Our previous investigations of cleavage in vivo of unmodified T4 DNA (24) as well as of a small plasmid (pBR322 [7]) suggested that recognition and cleavage by EndoII were dependent on helical structure. The helical structure of the recognition site may be influenced by factors outside the consensus sequence and may, in turn, influence the tracking of a processive enzyme along the DNA molecule.However, several features of T4 EndoII ...

Tumor overexpressed gene (TOG) protein, encoded by cytoskeleton-associated protein CKAP5, is a microtubule-associated protein that binds to heterogeneous nuclear ribonucleoprotein (hnRNP) A2. hnRNP A2 is an RNA trafficking factor that associates with myelin basic protein (MBP) mRNA. In oligodendrocytes, TOG, hnRNP A2, and MBP mRNA colocalize in granules that assemble in the perikaryon and are transported to the peripheral network of processes that extends from it. MBP accumulates preferentially in the membrane of the medial and distal portions of these cellular processes. MBP expression was reduced when TOG level was lowered by short-hairpin (sh) RNA. The reduction in TOG did not affect overall cell morphology or the assembly, transport, localization, or number of MBP mRNAcontaining granules. Reduced levels of TOG did not affect another oligodendrocyte-specific component, myelin oligodendrocyte glycoprotein, which is expressed at the same time as MBP but translated from mRNA localized in the cell body. Expression in a neural cell line of a green fluorescent protein (GFP)-MBP fusion protein derived from a construct containing GFP and the full-length cDNA for the rat 14 kDa MBP was reduced when TOG level was lowered by shRNA treatment. Expression of GFP, derived from GFP mRNA containing the hnRNP A2 binding element of MBP mRNA, was similarly reduced in cells with low TOG levels. These data indicate that TOG is necessary for efficient translation of MBP mRNA and suggest that this role is mediated by its interaction with hnRNP A2.