A novel family of nuclear transport receptors mediates the export of messenger RNA to the cytoplasm

Fully processed mRNAs are exported to the cytoplasm where they direct protein synthesis. A general feature of mRNA export is that it is an active, receptor-mediated process. The mRNA export receptors are thought to recognize and bind to the mRNA-export cargoes either directly or indirectly (via adaptor proteins) and facilitate their translocation across the central channel of the nuclear pore complex (NPC). On the cytoplasmic side of the NPC, the exported mRNA is released and the receptor returns to the nucleoplasm, without the cargo, to initiate additional rounds of export. Recent, studies in yeast and in higher eukaryotes have led to the elucidation of an evolutionarily conserved pathway for the export of bulk mRNA to the cytoplasm.     

The human tap nuclear RNA export factor contains a novel transportin-dependent nuclear localization signal that lacks nuclear export signal function.

The human Tap protein mediates the sequence-specific nuclear export of RNAs containing the constitutive transport element and is likely also critical for general mRNA export. Here, we demonstrate that a previously defined arginine-rich nuclear localization signal (NLS) present in Tap acts exclusively via the transportin import factor. Previously, transportin has been shown to mediate the nuclear import of several heterogeneous nuclear ribonucleoproteins, including heterogeneous nuclear ribonucleoprotein (hnRNP) A1, by binding to a sequence element termed M9. Although the Tap NLS and the hnRNP A1 M9 element are shown to compete for transportin binding, they show no sequence homology, and the Tap NLS does not conform to the recently defined M9 consensus. The Tap NLS also differs from M9 in that only the latter is able to act as a nuclear export signal. The Tap NLS is therefore the first member of a novel class of transportin-specific NLSs that lack nuclear export signal function.     

Identification of a 110 000 molecular weight protein associated with heterogeneous nuclear RNA and messenger RNA in rat liver cells

The composition of proteins associated with heterogeneous nuclear RNA (hnRNA) and polyribosomal messenger RNA (mRNA) in rat liver cells has been studied using sodium dodecylsulfate (SDS)-plate gel electrophoresis. The nuclear RNP was isolated as the 30–40 S monomer by means of several different procedures including extraction of nuclei at 0 and 25 °C and ultrasonic treatment. These preparations were shown to contain the same set of specific proteins when analysed electrophoretically. Dissociation of free polyribosomes was accomplished in the presence of either EDTA or puromycin at high ionic strength and the mRNP separated on columns of oligo(dT) cellulose. Two to three proteins with identical molecular weights were identified in the SDS band patterns of both hnRNP and mRNP; in particular a 110000 D double-band was most conspicuous in both band patterns.     

The synthesis and processing of a nuclear RNA precursor to rat pregrowth hormone messenger RNA.

A recombinant DNA plasmid, pBR322-GH1, which contains about 80% of the sequences of rat pregrowth hormone (pGH) mRNA, allowed an analysis of nuclear RNA from GH3 cells for possible precursors of cytoplasmic pGH mRNA. A single 20-22S RNA SPECIES ABOUT 2-3 TIMes larger than pGH mRNA was detected in nuclear RNA from GH3 cells labeled for 5 min. with 3H-uridine. After longer label times a 12S RNA indistinguishable in size from cytoplasmic 12S pGH mRNA became the predominant labeled RNA complementary to the plasmid pBR322-GH1. Both of these nuclear RNA species contained poly (A). Kinetic analysis of the labeling of nuclear and cytoplasmic pGH mRNA sequences showed that the 20S and 12S nuclear RNA molecules were labeled before significant labeling of cytoplasmic pGH mRNA was detected, and also indicated that there is complete conservation of nuclear pGH mRNA sequences in the production of cytoplasmic pGH mRNA. These results indicate that cytoplasmic pGH mRNA is generated by nuclear processing of a larger nuclear RNA molecule.     

Delayed processing/export of messenger RNA under conditions of reduced protein synthesis

The rates of processing and export of a variety of nuclear RNA species into the cytoplasmic compartment were studied by determining the rates of incorporation of tritiated uridine into nuclear and cytoplasmic RNA species. In exponentially growing cells, the rates of nuclear processing/export varied by more than a factor of ten for the six different mRNA species that were examined. Differences in the rates did not appear to be correlated with either the number or the sizes of introns in the genes for the RNA species. When cells were maintained under conditions of reduced protein synthesis (starvation for isoleucine and glutamine or exposure to cycloheximide), the processing rates for each species decreased by a factor of about 3. The decrease was not caused by the inability of hnRNA to associate with proteins, since the nuclear RNP distribution appeared normal in amino acid-starved cells.     

Methylation of late adenovirus 2 nuclear and messenger RNA.

Methylation of adenovirus 2 (Ad 2) late RNA was studied. RNA was double-labeled with [³H-methyl]-methionine and [¹⁴C]-uridine 15–20 h postinfection. Nuclear RNA (rRNA) and cytoplasmic RNA (mRNA) was extracted, and fractionated into polyA(+) and (?) molecules using poly(U)-Sepharose. Ad 2 specific RNA was purified by 2 cycles of hybridization to and elution from Ad 2 DNA immobilized on filters. The Ad 2 polyA(+) and (?) nRNA and mRNA fractions had the same ${}^3\text{H}{}^{14}\text{C}$ ratios, and were estimated to contain a minimum of 1.4 methylated nucleotides per 1000 bases. Viral RNA was digested with RNase T₂ and chromatographed on DEAE-Sephadex in 7 M urea at pH 7.6. All four Ad RNA fractions contained methylated constituents consistent with: (1) two classes of methylated “capped” 5′-termini with general structures m⁷ GpppN^mpNp and m⁷ GpppN^mpN^mpNp; (2) internal base methylations; (3) minor amounts of internal ribose 2′-0-methylations. Two classes of 5′-termini have previously been reported for animal cell mRNA, but not for mRNA from a variety of viruses. Internal methylations may be unique to RNA molecules transcribed in the nucleus, since they have not been found in RNA from cytoplasmic viruses. No gross differences were observed in the DEAE-Sephadex elution profiles of the methylated constituents of the four types of Ad 2 RNA. These results suggest that the majority of methylation events occur in the nucleus, and raise the possibility that Ad 2 methylated late nRNA may differ significantly from SV40 late nRNA (Lavi, S., and Shatkin, A.J. (1975) Proc. Natl. Acad. Sci. USA $\text{72}$, 2012–2016).     

The highly structured encapsidation signal of MuLV RNA is involved in the nuclear export of its unspliced RNA

p27Kip1 (p27) influences cell division by regulating nuclear cyclin-dependent kinases. Before binding, p27 is at least partially disordered and folds upon binding its Cdk/cyclin targets. 30-40% of human proteins, including p27, are predicted to contain disordered segments, and have been termed intrinsically unstructured proteins (IUPs). Unfortunately, the inherent dynamics of IUPs hamper detailed analysis of their structure/function relationships. Here, we describe the use of molecular dynamics (MD) computations and solution NMR spectroscopy to reveal that several segments of the p27 kinase inhibitory domain (p27-KID), in addition to the previously characterized helical segment, exist as highly populated, intrinsically folded structural units (IFSUs). Several IFSUs resemble structural features of bound p27-KID, while another exhibits alternative conformations. Interestingly, the highly conserved, specificity determining segment of p27 is shown to be highly disordered. Elucidation of IFSUs within p27-KID allows consideration of their influences on the thermodynamics and kinetics of Cdk/cyclin binding. The degree to which IFSUs are populated within p27-KID is surprising and suggests that other putative IUPs contain IFSUs that may be studied using similar techniques.