mRNA export: travelling with DEAD box proteins.

Recent studies have shown that the putative RNA helicase protein UAP56 and its yeast homologue Sub2p are not only involved in pre-mRNA splicing but also required for the export of mRNA out of the nucleus, even if the mRNA is encoded by an intron-less gene.

Nuclear export of mRNA.

Export of mRNA through nuclear pore complexes (NPC) is preceded by multiple and well coordinated processing steps, resulting in the formation of an export competent ribonucleoprotein complex (mRNP). Numerous factors involved in the translocation of the mRNP through the NPC and its release into the cytoplasm have been isolated mainly through genetic approaches in yeast, and putative functional homologues have been identified in metazoan systems. Understanding the mechanism of mRNA export relies, in part, on the functional characterization of these factors and the establishment of a complete network of molecular interactions. Here we summarize recent progress in the characterization of yeast and mammalian components implicated in the export of an mRNA from the nucleus to the cytoplasm.

The yeast hnRNP-Like proteins Yra1p and Yra2p participate in mRNA export through interaction with Mex67p.

Yra1p is an essential nuclear protein which belongs to the evolutionarily conserved REF (RNA and export factor binding proteins) family of hnRNP-like proteins. Yra1p contributes to mRNA export in vivo and directly interacts with RNA and the shuttling mRNP export receptor Mex67p in vitro. Here we describe a second nonessential Saccharomyces cerevisiae family member, called Yra2p, which is able to complement a YRA1 deletion when overexpressed. Like other REF proteins, Yra1p and Yra2p consist of two highly conserved N- and C-terminal boxes and a central RNP-like RNA-binding domain (RBD). These conserved regions are separated by two more variable regions, N-vr and C-vr. Surprisingly, the deletion of a single conserved box or the deletion of the RBD in Yra1p does not affect viability. Consistently, neither the conserved N and C boxes nor the RBD is required for Mex67p and RNA binding in vitro. Instead, the N-vr and C-vr regions both interact with Mex67p and RNA. We further show that Yra1 deletion mutants which poorly interact with Mex67p in vitro affect the association of Mex67p with mRNP complexes in vivo and are paralleled by poly(A)(+) RNA export defects. These observations support the idea that Yra1p promotes mRNA export by facilitating the recruitment of Mex67p to the mRNP.

Yeast Ran-binding protein 1 (Yrb1) shuttles between the nucleus and cytoplasm and is exported from the nucleus via a CRM1 (XPO1)-dependent pathway.

The RanGTP-binding protein RanBP1, which is located in the cytoplasm, has been implicated in release of nuclear export complexes from the cytoplasmic side of the nuclear pore complex. Here we show that Yrb1 (the yeast homolog of RanBP1) shuttles between the nucleus and the cytoplasm. Nuclear import of Yrb1 is a facilitated process that requires a short basic sequence within the Ran-binding domain (RBD). By contrast, nuclear export of Yrb1 requires an intact RBD, which forms a ternary complex with the Xpo1 (Crm1) NES receptor in the presence of RanGTP. Nuclear export of Yrb1, however, is insensitive towards leptomycin B, suggesting a novel type of substrate recognition between Yrb1 and Xpo1. Taken together, these data suggest that ongoing nuclear import and export is an important feature of Yrb1 function in vivo.

REF, an evolutionary conserved family of hnRNP-like proteins, interacts with TAP/Mex67p and participates in mRNA nuclear export.

Vertebrate TAP and its yeast ortholog Mex67p are involved in the export of messenger RNAs from the nucleus. TAP has also been implicated in the export of simian type D viral RNAs bearing the constitutive transport element (CTE). Although TAP directly interacts with CTE-bearing RNAs, the mode of interaction of TAP/Mex67p with cellular mRNAs is different from that with the CTE RNA and is likely to be mediated by protein-protein interactions. Here we show that Mex67p directly interacts with Yra1p, an essential yeast hnRNP-like protein. This interaction is evolutionarily conserved as Yra1p also interacts with TAP. Conditional expression in yeast cells implicates Yra1 p in the export of cellular mRNAs. Database searches revealed that Yra1p belongs to an evolutionarily conserved family of hnRNP-like proteins having more than one member in Mus musculus, Xenopus laevis, Caenorhabditis elegans, and Schizosaccharomyces pombe and at least one member in several species including plants. The murine members of the family directly interact with TAP. Because members of this protein family are characterized by the presence of one RNP-motif RNA-binding domain and exhibit RNA-binding activity, we called these proteins REF-bps for RNA and export factor binding proteins. Thus, Yra1p and members of the REF family of hnRNP-like proteins may facilitate the interaction of TAP/Mex67p with cellular mRNAs.

The RNA export factor Gle1p is located on the cytoplasmic fibrils of the NPC and physically interacts with the FG-nucleoporin Rip1p, the DEAD-box protein Rat8p/Dbp5p and a new protein Ymr 255p.

Gle1p is an essential, nuclear pore complex (NPC)-associated RNA export factor. In a screen for high copy suppressors of a GLE1 mutant strain, we identified the FG-nucleoporin Rip1p and the DEAD-box protein Rat8p/Dbp5p, both of which have roles in RNA export; we also found Ymr255p/Gfd1p, a novel inessential protein. All three high copy suppressors interact with the C-terminal domain of Gle1p; immunoelectron microscopy localizations indicate that Gle1p, Rip1p and Rat8p/Dbp5p are present on the NPC cytoplasmic fibrils; Rip1p was also found within the nucleoplasm and on the nuclear baskets. In vivo localizations support the hypothesis that Rip1p contributes to the association of Gle1p with the pore and that Gle1p, in turn, provides a binding site for Rat8p/Dbp5p at the NPC. These data are consistent with the view that Gle1p, Rip1p, Rat8p/Dbp5p and Ymr255p/Gfd1p associate on the cytoplasmic side of the NPC to act in a terminal step of RNA export. We also describe a human functional homologue of Rip1p, called hCG1, which rescues Rip1p function in yeast, consistent with the evolutionary conservation of this NPC-associated protein.

The importin-beta family member Crm1p bridges the interaction between Rev and the nuclear pore complex during nuclear export.

BACKGROUND: The human immunodeficiency virus (HIV-1) uses the viral protein Rev to regulate gene expression by promoting the export of unspliced and partially spliced viral transcripts. Rev has been shown to function in a variety of organisms, including Saccharomyces cerevisiae. The export activity of Rev depends on a nuclear export signal (NES), which is believed to interact either directly or indirectly with the nuclear pore complex to carry out its export function. Crm1p is a member of the importin-beta protein family, other members of which are known to be directly involved in nuclear import. Crm1p has recently been shown to contribute to nuclear export in vertebrate systems. Here, we have studied this mechanism of nuclear to cytoplasmic transport. RESULTS: Viable mis-sense mutations in the CRM1 gene substantially reduced or eliminated the biological activity of Rev in S. cerevisiae, providing strong evidence that Crm1p also contributes to transport of Rev NES-containing proteins and ribonucleoproteins in this organism. Crm1p interacted with FG-repeat-containing nuclear pore proteins as well as Rev, and we have demonstrated that the previously described two-hybrid interaction between Rev and the yeast nuclear pore protein Rip1p is dependent on wild-type Crm1p. CONCLUSIONS: We conclude that Crm1p interacts with the Rev NES and nuclear pore proteins during delivery of cargo to the nuclear pore complex. Our findings also agree well with current experiments on Crm1p orthologs in Schizosaccharomyces pombe and in vertebrate systems.

The yeast nucleoporin rip1p contributes to multiple export pathways with no essential role for its FG-repeat region.

The FG-repeat domain of the yeast Rip1 protein (Rip1p) was identified initially as a possible target for the nuclear export signal (NES) of the HIV-1 Rev protein in a yeast two-hybrid assay. Rip1p is inessential, associated with nuclear pore complexes, and structurally related to the FG-nucleoporin family of pore proteins. It contributes to HIV-1 Rev-mediated RNA export and is also important for the export of heat shock RNAs at 42 degrees C. We show here that Rip1p is essential for the export of heat shock RNAs, and this function is fulfilled by the unique carboxyl terminus of Rip1p with no substantial contribution from the FG-repeat region. Genetic interactions between Rip1p and the RNA export mediator Gle1p are described, which support a role of the carboxyl terminus of Rip1p in poly(A)+ RNA export. Finally, this domain of Rip1p also contributes to Rev-mediated RNA export. The data suggest that Rip1p promotes the nuclear export of different classes of substrates by contributing to optimal pore function.

A dynamic in vivo view of the HIV-I Rev-RRE interaction.

The export of pre-mRNAs coding for the structural genes of the human immunodeficiency virus type I depends on the interaction of the Rev protein with a highly structured viral RNA sequence, the Rev-responsive element (RRE). To gain information about the structure of the RRE and the determinants of the in vivo RRE-Rev interaction, we have analyzed the structure of the 351 nt RRE RNA within living yeast (Saccharomyces cerevisiae) by dimethyl sulfate probing with or without Rev. The in vivo structure in the absence of Rev is generally similar to the previously established solution structure. In addition, we observe a single hypermethylated guanine residue (G128), located within the Rev high-affinity binding site, in vitro as well as in vivo. The important homopurine interaction between residues 129 and 106 is required for the hyperreactivity, confirming its biological relevance. Expression of wild-type Rev leads to a protection of this region and to modifications of the RRE structure: the high-affinity site becomes further structured, and Stem IIA is destabilized. High-level expression of the oligomerization-defective mutant M4 protein leads to the same protections without destabilization of Stem IIA. Taken together with other observations, the data suggest that Rev captures the unusual conformation of the high-affinity site, followed by additional changes in the structure of the RRE.

A role for nucleoporin FG repeat domains in export of human immunodeficiency virus type 1 Rev protein and RNA from the nucleus.

The human immunodeficiency virus type 1 Rev protein contains a nuclear export signal (NES) that is required for Rev-mediated RNA export in mammals as well as in the yeast Saccharomyces cerevisiae. The Rev NES has been shown to specifically interact with a human (hRIP/RAB1) and a yeast (yRip1p) protein in the two-hybrid assay. Both of these interacting proteins are related to FG nucleoporins on the basis of the presence of typical repeat motifs. This paper shows that Rev is able to interact with multiple FG repeat-containing nucleoporins from both S. cerevisiae and mammals; moreover, the ability of Rev NES mutants to interact with these FG nucleoporins parallels the ability of the mutants to promote RNA export in yeast and mammalian cells. The data also show that, after Xenopus oocyte nuclear injection, several FG nucleoporin repeat domains inhibit the export of both Rev protein and U small nuclear RNAs, suggesting that these nucleoporins participate in Rev-mediated and cellular RNA export. Interestingly, not all FG nucleoporin repeat domains produced the same pattern of RNA export inhibition. The results suggest that Rev and cellular mediators of RNA export can interact with multiple components of the nuclear pore complex during transport, analogous to the proposed mode of action of the nuclear protein import receptor.

The yeast splicing factor Mud13p is a commitment complex component and corresponds to CBP20, the small subunit of the nuclear cap-binding complex.

The mechanism by which pre-mRNAs are initially recognized by the splicing machinery is not well understood. In the yeast system, commitment complexes are the earliest identified splicing complexes. They contain pre-mRNA, U1 snRNP, and the splicing factor Mud2p and probably correspond to the mammalian E complexes, which contain pre-mRNA, U1 snRNP, and the splicing factor U2AF. To identify other yeast commitment complex components, we have characterized mutant strains that are synthetic lethal with viable U1 snRNA mutations. We report here that MUD13 is a nonessential gene that encodes the yeast homolog of CBP20, the small subunit of the vertebrate nuclear cap-binding complex (CBC). Characterization of splicing in the delta-MUD13 strain and extract indicates that Mud13p is a yeast splicing factor and is the second identified non-snRNP commitment complex component. The observations also suggest that CBC interacts with other commitment complex components as well as with the substrate cap. Taken together with the accompanying results for a mammalian system, our data indicate that cap-binding proteins as well as the pre-mRNA cap contribute to early steps in spliceosome assembly.

Light source-induced error in computer-assisted image analysis with a video-based system.

OBJECTIVE: We tested the hypothesis that two different light sources, an alternating current fluorescent viewbox and a direct current halogen viewbox, do not differ with respect to their ability to illuminate reproducibly a radiograph during image capture. STUDY DESIGN: Two radiographs were taken: one with four hydroxyapatite chips mounted against a dry mandible and one without the chips. They were digitally subtracted with a video-based imaging system. The procedure was repeated at different times. RESULTS: A statistically significant difference among optical density measurements was found when the alternating current fluorescent viewbox (p < 0.001) was used and was related to light intensity variation. Such effect was not observed with the direct current halogen viewbox (p = 0.873). CONCLUSION: Study design efficiency was increased by 212% with the use of the direct current halogen viewbox so that to detect a specified treatment effect with a given level of statistical confidence, the sample size has to be 2.12 times greater if the alternating current fluorescent viewbox is used.

Spatial consequences of defective processing of specific yeast mRNAs revealed by fluorescent in situ hybridization.

This work introduces the first use of fluorescent in situ hybridization (FISH) to detect the distribution of specific transcripts in Saccharomyces cerevisiae. We have applied this technique to analysis of reporter transcripts from a single, integrated copy, or multicopy plasmids. We have evaluated the effect of splice site deletions or the presence or absence of a terminator/cleavage site and demonstrated that both splicing and polyadenylation affect the export of these transcripts from the nucleus to the cytoplasm. Moreover, we show that the exported pre-mRNAs are substrates for nonsense codon-mediated decay through the UPF1 pathway. The work presented here demonstrates that the spatial distribution of transcripts will also be an important component of yeast RNA metabolism.

Identification of a novel nuclear pore-associated protein as a functional target of the HIV-1 Rev protein in yeast.

The HIV-1 Rev protein increases the cytoplasmic levels of unspliced and singly spliced target transcripts in metazoan systems. Based on experiments that indicate a similar function of Rev in the yeast S. cerevisiae, we have identified a yeast protein that interacts with the effector domain of Rev. The protein, Rip1p, is a novel small nucleoporin-like protein, some of which is associated with nuclear pores. Its closest known yeast relative is a nuclear pore component also implicated in mRNA transport from nucleus to cytoplasm. Analysis of strains that overexpress Rip1p or that are deleted for the RIP1 gene show that Rip1p is important for the effect of Rev on gene expression, indicating that the physical interaction is of functional significance in vivo. The results suggest that Rev directly promotes the cytoplasmic transport of suitable transcripts by targeting them to the nuclear pore.

RNA structural patterns and splicing: molecular basis for an RNA-based enhancer.

Efficient splicing of the 325-nt yeast (Saccharomyces cerevisiae) rp51b intron requires the presence of two short interacting sequences located 200 nt apart. We used the powerful technique of randomization-selection to probe the overall structure of the intron and to investigate its role in pre-mRNA splicing. We identified a number of alternative RNA-RNA interactions in the intron that promote efficient splicing, and we showed that similar base pairings can also improve splicing efficiency in artificially designed introns. Only a very limited amount of structural information is necessary to create or maintain such a mechanism. Our results suggest that the base pairing contributes transiently to the spliceosome assembly process, most likely by complementing interactions between splicing factors. We propose that splicing enhancement by structure represents a general mechanism operating in large yeast introns that evolutionarily preceded the protein-based splicing enhancers of higher eukaryotes.

A functional interaction between Rev and yeast pre-mRNA is related to splicing complex formation.

The HIV-1 Rev protein regulates viral gene expression by enhancing the export of partially spliced or nonspliced viral transcripts to the cytoplasm. This activity is mediated by the interaction of Rev with a highly structured viral RNA sequence, the Rev response element (RRE). In this paper, Rev regulation has been recapitulated in the yeast Saccharomyces cerevisiae. The data show that Rev enhances yeast pre-mRNA translation in an RRE-dependent manner and requires intact RNA binding and effector domains. In addition, reporter gene mutations in the 5' splice site and in the branch point are epistatic to and eliminate Rev responsiveness, i.e. some spliceosome assembly pathway steps precede a functional interaction with Rev. The results indicate that some of the Rev-interacting components are conserved between yeast and mammals and suggest that Rev acts on yeast pre-mRNA in the context of the spliceosome.

mRNA nuclear export.

The export of mRNA from the nucleus to the cytoplasm is an essential step in the expression of genetic information in eukaryotes. It is an energy-dependent process and involves transport across the nuclear pores. It requires both cis-acting ribonucleoprotein particle signals and specific trans-acting factors. Although much remains to be learned, recent information has begun to define this pathway at both the cellular and biochemical levels and indicates that it is used as a key regulatory step by several viruses.

La proteins from Xenopus laevis. cDNA cloning and developmental expression.

In mammalian nuclei, newly-synthesized RNA polymerase III transcripts are transiently associated with a phosphorylated polypeptide of approximately 50 kDa called the La protein. Here we provide evidence that the frog Xenopus laevis contains mRNAs for two highly related La proteins, each apparently encoded by a single gene. Both forms of the La protein contain the RNP-80 motif previously identified in many RNA binding proteins. The steady state levels of La mRNAs and protein are approximately constant in oocytes, eggs and embryos. This implies a progressive and severe decrease in these levels on a per cell basis during early development. In particular, neither the La mRNA nor protein level increases at the mid-blastula transition, the time when RNA polymerase III transcription first occurs during embryogenesis.

U1 small nuclear ribonucleoprotein particle-protein interactions are revealed in Saccharomyces cerevisiae by in vivo competition assays.

Two highly conserved regions of the 586-nucleotide yeast (Saccharomyces cerevisiae) U1 small nuclear RNA (snRNA) can be mutated or deleted with little or no effect on growth rate: the universally conserved loop II (corresponding to the metazoan A loop) and the yeast core region (X. Liao, L. Kretzner, B. Séraphin, and M. Rosbash, Genes Dev. 4:1766-1774, 1990). To examine the contribution of these regions to U1 small nuclear ribonucleoprotein particle (snRNP) activity, a competitor U1 gene, encoding a nonfunctional U1 snRNA molecule, was introduced into a number of strains carrying a U1 snRNA gene with loop II or yeast core mutations. The presence of the nonfunctional U1 gene lowered the growth rate of these mutant strains but not wild-type strains, consistent with the notion that mutant U1 RNAs are less active than wild-type U1 snRNAs. A detailed analysis of the U1 snRNA levels and half-lives in a number of merodiploid strains suggests that these mutant U1 snRNAs interact with U1 snRNP proteins less well than do their wild-type counterparts. Competition for protein factors during snRNP assembly could account for a number of previous observations in both yeast and mammalian cells.