Unwinding Of Double-stranded RNA Research Articles

Identification of natural Rutaecarpine as a potent tobacco mosaic virus (TMV) helicase candidate for managingintractable plant viral diseases.

Naturally occurring alkaloids are particularly suitable for use as pesticide precursors and further modifications due to their cost-effectiveness, unique mechanism of action, tolerable degradation, and environmental friendliness. The famous tobacco mosaic virus (TMV) is a persistent plant pathogenic virus that can parasitize many plants and severely reduce crop production. To treat TMV disease, TMV helicase acts as a crucial target by hydrolyzing adenosine triphosphate (ATP) to provide energy for double-stranded RNA unwinding. To seek novel framework alkaloid leads targeting TMV helicase, this work successfully established an efficient screening platform for TMV helicase inhibitors based on natural alkaloids. In vivo activity screening, enzyme activity detection, and binding assays showed that Rutaecarpine from Evodia rutaecarpa (Juss.) Benth exhibited excellent TMV helicase inhibitory properties [dissociation constant (Kd ) = 1.1 μm, half maximal inhibitory concentration (IC50 ) = 227.24 μm] and excellent anti-TMV ability. Molecular docking and dynamic simulations depicted that Rutaecarpine could stably bind in active pockets of helicase with low binding energy (ΔGbind = -17.8 kcal/mol) driven by hydrogen bonding and hydrophobic interactions. Given Rutaecarpine's laudable bioactivity and structural modifiability, it can serve as a privileged building block for further pesticide discovery.

P68 RNA Helicase (DDX5) Required for the Formation of Various Specific and Mature miRNA Active RISC Complexes.

DEAD-box RNA helicases catalyze the ATP-dependent unwinding of doublestranded RNA. In addition, they are required for protein displacement and remodelling of RNA or RNA/protein complexes. P68 RNA helicase regulates the alternative splicing of the important protooncogene H-Ras, and numerous studies have shown that p68 RNA helicase is probably involved in miRNA biogenesis, mainly through Drosha and RISC/DICER complexes. This study aimed to determine how p68 RNA helicase affects the activity of selected mature miRNAs, including miR-342, miR-330, miR-138 and miR-206, miR-126, and miR-335, and let-7a, which are known to be related to cancer processes. The miRNA levels were analyzed in stable HeLa cells containing p68 RNA helicase RNAi induced by doxycycline (DOX). Relevant results were repeated using transient transfection with pSuper/ pSuper-p68 RNA helicase RNAi to avoid DOX interference. Herein, we reported that p68 RNA helicase downregulation increases the accumulation of the mature miRNAs, such as miR-126, let-7a, miR-206, and miR-138. Interestingly, the accumulation of these mature miRNAs does not downregulate their known protein targets, thus suggesting that p68 RNA helicase is required for mature miRNA-active RISC complex activity. Furthermore, we demonstrated that this requirement is conserved, as drosophila p68 RNA helicase can complete the p68 RNA helicase depleted activity in human cells. Dicer and Drosha proteins are not affected by the downregulation of p68 RNA helicase despite the fact that Dicer is also localized in the nucleus when p68 RNA helicase activity is reduced.

DDX21 Interacts with WDR5 to Promote Colorectal Cancer Cell Proliferation by Activating CDK1 Expression.

DEAD-box RNA helicase 21 (DDX21), is a nucleolar protein harboring ATP-dependent double-stranded RNA unwinding activities, essential in rRNA processing and ribosome biogenesis. However, its role in colorectal cancer (CRC) progression remains unclear. In this study, we show that knockdown of DDX21 significantly inhibited CRC cell proliferation and blocked cell cycle at the G2/M phase. Gene profile analysis and ChIP assays revealed that DDX21 activated CDK1 gene expression through binding to the gene promoter. In addition, we found that DDX21 directly recruited WDR5 to enhance trimethylation of histone H3 on Lys 4 (H3K4me3) on the CDK1 promoter. Importantly, elevated expression of DDX21 in CRC patients was positively correlated with expression of CDK1, and these CRC patients had shorter overall survival. These findings reveal a critical novel role of DDX21 in transcriptional and epigenetic control of CRC cell proliferation. Taken together, this study uncovers that DDX21 interacted with WDR5 to promote colorectal cancer cell proliferation by activating CDK1 expression, suggesting that targeting DDX21 may be an alternative new strategy for CRC treatment.

Identification of RNA helicases in Medicago truncatula and their expression patterns under abiotic stress.

The online version contains supplementary material available at 10.1007/s12298-021-01087-y.

Assignment of the Ile, Leu, Val, Met and Ala methyl group resonances of the DEAD-box RNA helicase DbpA from E. coli

ATP-dependent DEAD-box helicases constitute one of the largest families of RNA helicases and are important regulators of most RNA-dependent cellular processes. The functional core of these enzymes consists of two RecA-like domains. Changes in the interdomain orientation of these domains upon ATP and RNA binding result in the unwinding of double-stranded RNA. The DEAD-box helicase DbpA from E. coli is involved in ribosome maturation. It possesses a C-terminal RNA recognition motif (RRM) in addition to the canonical RecA-like domains. The RRM recruits DbpA to nascent ribosomes by binding to hairpin 92 of the 23S rRNA. To follow the conformational changes of Dbpa during the catalytic cycle we initiated solution state NMR studies. We use a divide and conquer approach to obtain an almost complete resonance assignment of the isoleucine, leucine, valine, methionine and alanine methyl group signals of full length DbpA (49 kDa). In addition, we also report the backbone resonance assignments of two fragments of DbpA that were used in the course of the methyl group assignment. These assignments are the first step towards a better understanding of the molecular mechanism behind the ATP-dependent RNA unwinding process catalyzed by DEAD-box helicases.

The Human RNA Helicase DDX21 Presents a Dimerization Interface Necessary for Helicase Activity.

SummaryMembers of the DEAD-box helicase family are involved in all fundamental processes of RNA metabolism, and as such, their malfunction is associated with various diseases. Currently, whether and how oligomerization impacts their biochemical and biological functions is not well understood. In this work, we show that DDX21, a human DEAD-box helicase with RNA G-quadruplex resolving activity, is dimeric and that its oligomerization state influences its helicase activity. Solution small-angle X-ray scattering (SAXS) analysis uncovers a flexible multi-domain protein with a central dimerization domain. While the Arg/Gly rich C termini, rather than dimerization, are key to maintaining high affinity for RNA substrates, in vitro helicase assays indicate that an intact dimer is essential for both DDX21 ATP-dependent double-stranded RNA unwinding and ATP-independent G-quadruplex remodeling activities. Our results suggest that oligomerization plays a key role in regulating RNA DEAD-box helicase activity.

Theoretical Modeling of the RNA-Enhanced NTPase Activity of Dengue and Zika NS3 Helicases

The non-structural 3 helicase (NS3h) protein is a RNA helicase, encoded byFlaviviridae viruses, that plays a pivotal role during the replication of the viral genome. Energy released through the NTP hydrolysis reaction cycle powers the unidirectional translocation and unwinding of double-stranded RNA. Additionally, experiment has demonstrated that bound RNA enhances the hydrolysis activity by approximately 10-100 fold. Simulations of the dengue and Zika NS3h will be presented to discuss the RNA-induced enhancement of the NTP hydrolysis reaction cycle. As reported in our recent paper (Davidsonet al. PLOS Comput. Biol. 2018, 14 (4), e1006103), microsecond-long molecular dynamics simulations of dengue virus NS3h are coupled with electronic structure calculations of the hydrolysis active site to investigate this enhancement of activity. Results from these simulations reveal that bound RNA affects the positioning of active site residues and the hypothesized lytic water, leading to an estimated hydrolysis enhancement of 150 fold as calculated from the combined theoretical methods. Second-generation analyses will be presented that provide more details into the mechanistic origins of this calculated enhancement factor. Furthermore, MD simulations of the Zika virus NS3h have been used to test the hypotheses and conclusions drawn from our body of calculations of dengue NS3h. This combined study of dengue and Zika NS3h allows us to ask the important questions of “Are the simulations’ results consistent? Do results for one system adequately describe the results for another?” Results from both dengue and Zika simulations will be compared so as to answer these questions from which testable hypotheses and new experiments will be proposed.

Mechanism of ATP hydrolysis by the Zika virus helicase.

During its life cycle, Zika virus (ZIKV), an arthropod-borne flavivirus that is associated with Guillain-Barré syndrome and causes microencephaly in fetuses and newborn children, encodes a critical and indispensable helicase domain that has 5'-triphosphatase activity and performs ATP hydrolysis to generate energy and thus, sustains unwinding of double-stranded RNA during ZIKV genome replication. Of these processes, ATP hydrolysis represents the most basic event; however, its dynamic mechanisms remain largely unknown, impeding the further understanding of the function of ZIKV helicase and the ongoing anti-ZIKV drug design. In this work, we determined the crystal structure of ZIKV helicase in complex with ADP-AlF3-Mn2+ and ADP-Mn2+ separately. The structural analysis indicates that these structures represent the intermediate state and posthydrolysis state, respectively, of the ATP hydrolysis process of ZIKV helicase. These findings, together with our earlier work, which identified the prehydrolysis state of ZIKV helicase, lead to a proposal of the ATP hydrolysis cycle for ZIKV helicase. On this basis, we used site-directed mutagenesis combined with an enzymatic study to identify successfully residues that are critical for the ATPase activity of ZIKV helicase; this will provide new ideas to understand the function for the key enzyme of ZIKV.-Yang, X., Chen, C., Tian, H., Chi, H., Mu, Z., Zhang, T., Yang, K., Zhao, Q., Liu, X., Wang, Z., Ji, X., Yang, H. Mechanism of ATP hydrolysis by the Zika virus helicase.

DEAD-box RNA helicase domains exhibit a continuum between complete functional independence and high thermodynamic coupling in nucleotide and RNA duplex recognition.

DEAD-box helicases catalyze the non-processive unwinding of double-stranded RNA (dsRNA) at the expense of adenosine triphosphate (ATP) hydrolysis. Nucleotide and RNA binding and unwinding are mediated by the RecA domains of the helicase core, but their cooperation in these processes remains poorly understood. We therefore investigated dsRNA and nucleotide binding by the helicase cores and the isolated N- and C-terminal RecA domains (RecA_N, RecA_C) of the DEAD-box proteins Hera and YxiN by steady-state and time-resolved fluorescence methods. Both helicases bind nucleotides predominantly via RecA_N, in agreement with previous studies on Mss116, and with a universal, modular function of RecA_N in nucleotide recognition. In contrast, dsRNA recognition is different: Hera interacts with dsRNA in the absence of nucleotide, involving both RecA domains, whereas for YxiN neither RecA_N nor RecA_C binds dsRNA, and the complete core only interacts with dsRNA after nucleotide has been bound. DEAD-box proteins thus cover a continuum from complete functional independence of their domains, exemplified by Mss116, to various degrees of inter-domain cooperation in dsRNA binding. The different degrees of domain communication and of thermodynamic linkage between dsRNA and nucleotide binding have important implications on the mechanism of dsRNA unwinding, and may help direct RNA helicases to their respective cellular processes.

Functional Cross-talk between Distant Domains of Chikungunya Virus Non-structural Protein 2 Is Decisive for Its RNA-modulating Activity

Chikungunya virus (CHIKV) non-structural protein 2 (nsP2) is a multifunctional protein that is considered a master regulator of the viral life cycle and a main viral factor responsible for cytopathic effects and subversion of antiviral defense. The C-terminal part of nsP2 possesses protease activity, whereas the N-terminal part exhibits NTPase and RNA triphosphatase activity and is proposed to have helicase activity. Bioinformatics analysis classified CHIKV nsP2 into helicase superfamily 1. However, the biochemical significance of a coexistence of two functionally unrelated modules in this single protein remains unknown. In this study, recombinant nsP2 demonstrated unwinding of double-stranded RNA in a 5'-3' directionally biased manner and RNA strand annealing activity. Comparative analysis of NTPase and helicase activities of wild type nsP2 with enzymatic capabilities of different truncated or N-terminally extended variants of nsP2 revealed that the C-terminal part of the protein is indispensable for helicase functionality and presumably provides a platform for RNA binding, whereas the N-terminal-most region is apparently involved in obtaining a conformation of nsP2 that allows for its maximal enzymatic activities. The establishment of the protocols for the production of biochemically active CHIKV nsP2 and optimization of the parameters for helicase and NTPase assays are expected to provide the starting point for a further search of possibilities for therapeutic interventions to suppress alphaviral infections.

RNA Unwinding by NS3 Helicase: A Statistical Approach

The study of double-stranded RNA unwinding by helicases is a problem of basic scientific interest. One such example is provided by studies on the hepatitis C virus (HCV) NS3 helicase using single molecule mechanical experiments. HCV currently infects nearly 3% of the world population and NS3 is a protein essential for viral genome replication. The objective of this study is to model the RNA unwinding mechanism based on previously published data and study its characteristics and their dependence on force, ATP and NS3 protein concentration. In this work, RNA unwinding by NS3 helicase is hypothesized to occur in a series of discrete steps and the steps themselves occurring in accordance with an underlying point process. A point process driven change point model is employed to model the RNA unwinding mechanism. The results are in large agreement with findings in previous studies. A gamma distribution based renewal process was found to model well the point process that drives the unwinding mechanism. The analysis suggests that the periods of constant extension observed during NS3 activity can indeed be classified into pauses and subpauses and that each depend on the ATP concentration. The step size is independent of external factors and seems to have a median value of 11.37 base pairs. The steps themselves are composed of a number of substeps with an average of about 4 substeps per step and an average substep size of about 3.7 base pairs. An interesting finding pertains to the stepping velocity. Our analysis indicates that stepping velocity may be of two kinds- a low and a high velocity.

The mechanism of ATP-dependent RNA unwinding by DEAD box proteins

DEAD box proteins catalyze the ATP-dependent unwinding of double-stranded RNA (dsRNA). In addition, they facilitate protein displacement and remodeling of RNA or RNA/protein complexes. Their hallmark feature is local destabilization of RNA duplexes. Here, we summarize current data on the DEAD box protein mechanism and present a model for RNA unwinding that integrates recent data on the effect of ATP analogs and mutations on DEAD box protein activity. DEAD box proteins share a conserved helicase core with two flexibly linked RecA-like domains that contain all helicase signature motifs. Variable flanking regions contribute to substrate binding and modulate activity. In the presence of ATP and RNA, the helicase core adopts a compact, closed conformation with extensive interdomain contacts and high affinity for RNA. In the closed conformation, the RecA-like domains form a catalytic site for ATP hydrolysis and a continuous RNA binding site. A kink in the backbone of the bound RNA locally destabilizes the duplex. Rearrangement of this initial complex generates a hydrolysis- and unwinding-competent state. From this complex, the first RNA strand can dissociate. After ATP hydrolysis and phosphate release, the DEAD box protein returns to a low-affinity state for RNA. Dissociation of the second RNA strand and reopening of the cleft in the helicase core allow for further catalytic cycles.

Studies on three E. coli DEAD-box helicases point to an unwinding mechanism different from that of model DNA helicases.

DEAD-box proteins participate in various aspects of RNA metabolism in all organisms. These RNA-dependent ATPases are usually regarded as double-stranded RNA unwinding enzymes, though in vitro this activity has only been demonstrated for a subset of them. Given their high biological specificity, their equivocal unwinding activity may reflect the noncognate character of the substrates used in vitro. Here, we pinpoint other reasons for this elusiveness. We have compared the ATPase and helicase activities of three E. coli DEAD-box proteins, CsdA, RhlE and SrmB. Whereas the ATPase activity of all proteins is stimulated (albeit to various degree) by long RNAs, only RhlE is stimulated by short oligoribonucleotides. Consistently, all three proteins can unwind RNA duplexes with long single-stranded extensions, but only RhlE is effective when extensions are short or absent. Another critical constraint concerns the length of the duplex region: in the case of RhlE, the ratio (duplex unwound)/(ATP hydrolyzed) drops 1000-fold upon going from 11 to 14 base pairs, indicating a low processivity. Remarkably, allowing for these constraints, all three proteins can unwind substrates with either 5' or 3' extensions (or no extension in the case of RhlE). This behavior, which contrasts with that of well studied SF1 DNA helicases, is discussed in the light of available structural and biochemical data.

DEAD Box RhlB RNA Helicase Physically Associates with Exoribonuclease PNPase to Degrade Double-stranded RNA Independent of the Degradosome-assembling Region of RNase E

The Escherichia coli RNA degradosome is a multicomponent ribonucleolytic complex consisting of three major proteins that assemble on a scaffold provided by the C-terminal region of the endonuclease, RNase E. Using an E. coli two-hybrid system, together with BIAcore apparatus, we investigated the ability of three proteins, polynucleotide phosphorylase (PNPase), RhlB RNA helicase, and enolase, a glycolytic protein, to interact physically and functionally independently of RNase E. Here we report that Rh1B can physically bind to PNPase, both in vitro and in vivo, and can also form homodimers with itself. However, binding of RhlB or PNPase to enolase was not detected under the same conditions. BIAcore analysis revealed real-time, direct binding for bimolecular interactions between Rh1B units and for the RhlB interaction with PNPase. Furthermore, in the absence of RNase E, purified RhlB can carry out ATP-dependent unwinding of double-stranded RNA and consequently modulate degradation of double-stranded RNA together with the exonuclease activity of PNPase. These results provide evidence for the first time that both functional and physical interactions of individual degradosome protein components can occur in the absence of RNase E and raise the prospect that the RNase E-independent complexes of RhlB RNA helicase and PNPase, detected in vivo, may constitute mini-machines that assist in the degradation of duplex RNA in structures physically distinct from multicomponent RNA degradosomes.

Solution structure and backbone dynamics of an engineered arginine-rich subdomain 2 of the hepatitis C virus NS3 RNA helicase

The NS3 protein of the hepatitis C virus (HCV) is a 631 amino acid residue bifunctional enzyme with a serine protease localized to the N-terminal 181 residues and an RNA helicase located in the C-terminal 450 residues. The HCV NS3 RNA helicase consists of three well-defined subdomains which all contribute to its helicase activity. The second subdomain of the HCV helicase is flexibly linked to the remainder of the NS3 protein and could undergo rigid-body movements during the unwinding of double-stranded RNA. It also contains several motifs that are implicated in RNA binding and in coupling NTP hydrolysis to nucleic acid unwinding and translocation. As part of our efforts to use NMR techniques to assist in deciphering the enzyme’s structure-function relationships and developing specific small molecule inhibitors, we have determined the solution structure of an engineered subdomain 2 of the NS3 RNA helicase of HCV, d2Δ-HCVh, and studied the backbone dynamics of this protein by 15N-relaxation experiments using a model-free approach. The NMR studies on this 142-residue construct reveal that overall subdomain 2 of the HCV helicase is globular and well structured in solution even in the absence of the remaining parts of the NS3 protein. Its solution structure is very similar to the corresponding parts in the X-ray structures of the HCV NS3 helicase domain and intact bifunctional HCV NS3 protein. Slow hydrogen-deuterium exchange rates map to a well-structured, stable hydrophobic core region away from the subdomain interfaces. In contrast, the regions facing the subdomain interfaces in the HCV NS3 helicase domain are less well structured in d2Δ-HCVh, show fast hydrogen-deuterium exchange rates, and the analysis of the dynamic properties of d2Δ-HCVh reveals that these regions of the protein show distinct dynamical features. In particular, residues in motif V, which may be involved in transducing allosteric effects of nucleotide binding and hydrolysis on RNA binding, exhibit slow conformational exchange on the milli- to microsecond time-scale. The intrinsic conformational flexibility of this loop region may facilitate conformational changes required for helicase function.

Cloning and characterization of a DEAD box RNA helicase from the viable seedlings of aged mung bean.

Seeds stored under adverse conditions will reduce the viability of germination as a result of induced aging. We have established a procedure to induce accelerated aging for studying the process of aging in mung bean (Vigna radiata) seeds at the molecular level. A full-length cDNA was isolated from acceleratedly aged mung bean seedlings. The cDNA, VrRH1 (Vigna radiata RNA helicase 1), contains an open reading frame of 2139 bp encoding a protein of 713 amino acids. VrRHI has seven highly conserved motifs including the DEAD box as in the case of other plant RNA helicases. VrRHI was sub-cloned into an expression vector pET-28b (+), over-expressed in Escherichia coli BL 21 and purified by a Ni2+-agarose column. The expressed protein showed double-stranded RNA unwinding and ATPase activities. Either ATP or dATP is required for the unwinding activity, indicating that VrRHI is an ATP/dATP-dependent RNA helicase. Northern blot analysis showed the presence of mRNAs hybridized with a full-length cDNA fragment of VrRHI (VrRH transcripts) in mung bean seeds that were imbibed for 16 to 32 h after accelerated aging treatment. The amount of these mRNAs reached a maximum in 24 h imbibed seeds after the treatment. The accumulation of VrRH transcripts was shown to lead to the appearance of 25S and 18S rRNAs in the imbibed aging mung bean seeds. The results suggest that VrRHI may play a role in the viability of mung bean seeds.

Sequence analysis of a 24-kb contiguous genomic region at the Arabidopsis thaliana PFL locus on chromosome 1

As part of the European Union program of European Scientist Sequencing Arabidopsis (ESSA), the DNA sequence of a 24.053-bp insert of cosmid clone CC17J13 was determined. The cosmid is located on chromosome 1 at the PFL locus (position 30 cM). Analysis of the sequence and comparison to public databases predicts seven genes in this area, thus approximately one gene every 3.3 kb. Three cDNAs corresponding to genes in this region were also sequenced. The homologies and/or possible functions of the (putative) genes are discussed. Proteins encoded by genes in this region include a polyadenylate-binding protein (PAB-3) and a GTP-binding protein (Rab7) as well as a novel protein, possibly involved in double-stranded RNA unwinding and apoptosis. Intriguingly, the gene encoding the PAB-3 protein, which is very specifically expressed, is flanked by putative matrix attachment regions.

Characterization of the autoantigen La (SS-B) as a dsRNA unwinding enzyme.

During the analysis of the La (SS-B) autoantigen for catalytic activities an ATP-dependent double-stranded RNA unwinding activity was detected. Both native and recombinant La proteins from different species displayed this activity, which could be inhibited by monospecific anti-La antibodies. La protein was able to melt dsRNA substrates with either two 3'-overhangs or a single 3'- and a 5'-overhang. Double-stranded RNAs with two 5'-overhangs were not unwound, indicating that at least one 3'-overhang is required for unwinding. Sequence elements of the La protein that might be involved in dsRNA unwinding, such as an evolutionarily conserved putative ATP-binding motif and an element that is homologous to the double-stranded RNA binding protein kinase PKR, are discussed.

Translation Initiation Factor 4A from Saccharomyces cerevisiae: Analysis of Residues Conserved in the D-E-A-D Family of RNA Helicases

The eukaryotic translation initiation factor 4A (eIF-4A) possesses an in vitro helicase activity that allows the unwinding of double-stranded RNA. This activity is dependent on ATP hydrolysis and the presence of another translation initiation factor, eIF-4B. These two initiation factors are thought to unwind mRNA secondary structures in preparation for ribosome binding and initiation of translation. To further characterize the function of eIF-4A in cellular translation and its interaction with other elements of the translation machinery, we have isolated mutations in the TIF1 and TIF2 genes encoding eIF-4A in Saccharomyces cerevisiae. We show that three highly conserved domains of the D-E-A-D protein family, encoding eIF-4A and other RNA helicases, are essential for protein function. Only in rare cases could we make a conservative substitution without affecting cell growth. The mutants show a clear correlation between their growth and in vivo translation rates. One mutation that results in a temperature-sensitive phenotype reveals an immediate decrease in translation activity following a shift to the nonpermissive temperature. These in vivo results confirm previous in vitro data demonstrating an absolute dependence of translation on the TIF1 and TIF2 gene products.

Translation initiation factor 4A from Saccharomyces cerevisiae: analysis of residues conserved in the D-E-A-D family of RNA helicases.

The eukaryotic translation initiation factor 4A (eIF-4A) possesses an in vitro helicase activity that allows the unwinding of double-stranded RNA. This activity is dependent on ATP hydrolysis and the presence of another translation initiation factor, eIF-4B. These two initiation factors are thought to unwind mRNA secondary structures in preparation for ribosome binding and initiation of translation. To further characterize the function of eIF-4A in cellular translation and its interaction with other elements of the translation machinery, we have isolated mutations in the TIF1 and TIF2 genes encoding eIF-4A in Saccharomyces cerevisiae. We show that three highly conserved domains of the D-E-A-D protein family, encoding eIF-4A and other RNA helicases, are essential for protein function. Only in rare cases could we make a conservative substitution without affecting cell growth. The mutants show a clear correlation between their growth and in vivo translation rates. One mutation that results in a temperature-sensitive phenotype reveals an immediate decrease in translation activity following a shift to the nonpermissive temperature. These in vivo results confirm previous in vitro data demonstrating an absolute dependence of translation on the TIF1 and TIF2 gene products.