Biosynthesis of reovirus-specified polypeptides: Initiation of reovirus messenger RNA translation in vitro and identification of methionyl-x initiation peptides

The initiation of reovirus messenger RNA-directed protein synthesis in vitro was investigated in a cell-free protein synthesizing system prepared from Krebs ascites tumor cells. The principal translation products of the mixture of 10 reovirus mRNA species transcribed in vitro by reovirus cores were polypeptides μ₀, μ₁, σ_2a, and σ₃. Translation could be initiated with formylmethionine transferred from rat liver methionyl-tRNA formylated by Escherichia coli formyltransferase with 10-formyltetrahydrafolate as the formyl donor. Formylmethionine incorporation was complete within 10 to 15 min and was inhibited by aurin tricarboxylic acid and pactamycin; by contrast, incorporation of methionine and leucine continued for 30 to 60 min.The identification of the amino acids at the amino termini of polypeptides μ₀, μ₁, σ_2a, and σ₃ synthesized in vitro was elucidated. Protein synthesis was carried out in the presence of rat liver formylmethionyl-tRNA and various groups of radioactively labeled amino acids. The viral polypeptides that were synthesized were isolated by urea-SDS-polyacrylamide gel electrophoresis and digested with pronase. N-Formylmethionylcontaining peptides were then separated from other peptides by fractionation on Dowex-50 and hydrolyzed with acid. The radioactive amino acids that were liberated were then identified by two-dimensional thin-layer chromatography. The following amino terminal assignments were elucidated: μ₀, (N-formyl)methionyl-valyl-(proline); μ1, (N-formyl)methionyl-leucyl-valine; σ_2a, (N-formyl)methionyl-threonyl-valine; and σ₃, (N-formyl)methionyl-valyl-tyrosyl-(proline).No evidence was obtained for amino terminal acetylation or formylation of reovirus-specific protein synthesized in vitro in the absence of exogenously added formylmethionyl-transfer RNA.     

The relative translation efficiencies of reovirus messenger RNAs

The relative translation efficiencies of reovirus messenger RNAs were measured by infecting BHK cells with serotype 1, 2, or 3 virus and measuring the rate of formation of each viral protein species and the amount of each viral single-stranded (ss) RNA species present in the infected cells throughout the multiplication cycle. The translation efficiency of each ssRNA species was then calculated as a percentage of the species that was translated most efficiently. The relative translation efficiencies of the various ssRNA species did not change significantly during the multiplication cycle and cognate mRNA species of serotype 1, 2, and 3 virus were generally translated with similar efficiencies. However, the relative translation efficiencies of the individual ssRNA species differed greatly. The most efficiently translated ssRNA species was usually species s4, followed by species m2 which, on average, was translated about two-thirds as efficiently as species s4. Species s2 and s3 were translated slightly less than one-half as efficiently as species s4; species m3, l2, and l3 one-quarter to one-third as efficiently; and species s1 one-twentieth to one-tenth as efficiently. Finally, species l1 and m1 were translated very inefficiently under all conditions; their translation efficiencies were usually no more than 1% of that of species s4.     

Efficient extraction and translation of Plasmodium falciparum messenger RNA

A convenient method was employed for the efficient extraction of total RNA from Plasmodium falciparum. By depleting the total RNA of tRNA, it was shown that P. falciparum or P. lophurae tRNAs markedly stimulate the translation of P. falciparum mRNA in the rabbit reticulocyte cell-free system. Analysis of the cell-free products revealed the presence of proteins well over 200 000 molecular weight, with the majority of polypeptides having high molecular weights. Thus, this system can now be used to isolate and characterize specific mRNA molecules from P. falciparum.     

Noncanonical repression of translation initiation through small RNA recruitment of the RNA chaperone Hfq

The RNA chaperone Hfq is mostly known to help small regulatory RNAs (sRNAs) interact with target mRNAs to block initiating ribosomes. In this model, whereas the sRNA is directly competing with initiating 30S ribosomal subunits, Hfq plays only an indirect role, allowing optimal sRNA-mRNA pairing. Here we report that Hfq is recruited by a sRNA, Spot42, to bind to a precise AU-rich region in the vicinity of the translation initiation region (TIR) of sdhC mRNA and competes directly with 30S ribosomal subunits. We show that the sRNA Spot42 binds sdhC too far upstream of the TIR to directly repress translation initiation in vitro and in vivo. Contrary to the canonical model of sRNA regulation, this suggests a new mechanism where Hfq is directly involved in the translational repression of the target mRNA and where the sRNA acts only as a recruitment factor.     

Isolation and in vitro translation of messenger RNA from the American cockroach

Total RNA was extracted from the whole body of American cockroach Periplaneta americana) using chaotropic salt guanidine isothiocyanate in the presence of 2-mercaptoethanol (2-ME). Polyadenylated mRNA was isolated by oligothymidylic acidcellulose chromatography and mRNA was translated using a rabbit reticulocyte lysate system. The translation, as judged by the incorporation of-S-tnethionine, was obtained with poly(A)‘*’RNA, where an approximately 9-5-fold increase in label incorporation over control was achieved. Analysis of translation products by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in combination with autoradiography showed that many proteins with apparent molecular weights ranging from 12 to 200 kD were synthesized, and no labelled proteins were found with negative RNA control and poly(A) RNA. Immunoprecipitation studies performed using polyclonal and monoclonal antibodies revealed that synthesized proteins of MW 90, 78, 72, 49, 45, and 26 kD corresponded with previously identified principal and major allergens of American cockroach from our laboratory. In addition, the allergenicity of the translation mixtures was also confirmed by fluoroallergosorbent test (FAST) inhibition studies with IgE antibodies of human reaginic serum pool.     

Mechanism of interferon action studies on the mechanism of interferon-mediated inhibition of reovirus messenger RNA translation in cell-free protein synthesis systems from mouse ascites tumor cells

The biochemical mechanism of the interferon-mediated inhibition of reovirus messenger RNA translation in cell-free extracts prepared from mouse ascites tumor cells was investigated. The following results were obtained: (1) Cell-free extracts from interferon-treated cells catalyze the formation of methionyl-X initiation dipeptides in response to reovirus mRNA at a rate and extent only slightly less than that of extracts from untreated cells. Under conditions where the overall translation in vitro of reovirus mRNA is inhibited by 75% or more, the formation of methionyl-X initiation dipeptides is inhibited much less, about 30%. (2) Neither methionyl-X initiation dipeptide formation nor overall translation of reovirus mRNA is significantly affected by the addition of either S-adenosyl-l-methionine or S-adenosyl-l-homocysteine to cell-free systems prepared from either interferon-treated or untreated cells. (3) Reovirus mRNA synthesized in vitro in the presence of S-adenosyl-l-methionine is translated slightly more efficiently than either reovirus mRNA synthesized in the presence of S-adenosyl-l-homocysteine or reovirus mRNA synthesized in the absence of both S-adenosyl-l-methionine and S-adenosyl-l-homocysteine by cell-free systems prepared from untreated ascites cells; by contrast, all of the reovirus mRNA preparations were translated very poorly by cell-free systems prepared from interferon-treated cells. (4) Exogenously added mouse ascites cell transfer RNA partially reverses the inhibition of viral mRNA translation in vitro catalyzed by cell-free systems prepared from interferon-treated cells in response to reovirus mRNA synthesized in the absence or in the presence of either S-adenosyl-l-methionine or S-adenosyl-l-homocysteine. Yeast and rat liver tRNA as well as tRNA from two prokaryotes, Escherichia coli and Streptococcus faecalis, do not significantly stimulate viral mRNA translation in the interferon-treated cell-free system. Translation catalyzed by untreated control extracts is not dependent upon the addition of tRNA. (5) Reovirus [³²P]mRNA recovered from interferon-treated cell-free protein synthesis systems after 15-min incubation possesses a profile similar to that of mRNA recovered from untreated control systems when analyzed by polyacrylamide-agarose-urea gel electrophoresis. These results indicate that the interferon-mediated inhibition of viral mRNA translation in vitro is exerted at a step of polypeptide chain biosynthesis which is subsequent to formation of the first peptide bond and which either directly or indirectly involves the participation of transfer RNA.     

In vitro translation of messenger RNA from the house dust mite Dermatophagoides pteronyssinus

RNA was extracted from the house dust mite Dermatophagoides pteronyssinus and a polyadenylated [poly(A)+] and non-polyadenylated [poly(A)-] mRNA isolated by oligothymidylic acid-cellulose chromatography. Both preparations were translated in vitro using a cell-free rabbit reticulocyte system. The relative incorporation of 35S-methionine into trichloroacetic acid-precipitable translation products obtained using poly(A)+ and poly(A)- mRNA was 21- and 1.3-fold, respectively, when compared with control translations performed without added mRNA. Sodium dodecylsulphate-polyacrylamide gel electrophoretic analysis of the translation products in combination with autoradiography showed that many proteins with apparent molecular weights in the range 14,000- greater than 67,000 daltons were synthesized. Immunoprecipitation studies performed using a sheep anti-whole mite antiserum showed that several of the synthesized proteins corresponded with mite antigens. These findings were confirmed by coprecipitation studies using crossed immunoelectrophoresis (CIE). A comparison of the CIE data with those obtained with crossed radio-immunoelectrophoresis showed that 6 of the 9 allergens previously described in our laboratory were synthesized, namely Der p I, Dpt 1, 3, 4, 8 and 17. The allergenicity of the lysates was also confirmed by immunoprecipitation studies using a specific anti-human IgE reagent.     

Analysis of a non-lethal biallelic frameshift mutation in ZMPSTE24 reveals utilization of alternative translation initiation codons

Restrictive dermopathy (RD) is a lethal condition caused by biallelic loss-of-function mutations in ZMPSTE24, whereas mutations preserving residual enzymatic activity of the ZMPSTE24 protein lead to the milder mandibuloacral dysplasia with type B lipodystrophy (MADB) phenotype. Remarkably, we identified a homozygous, presumably loss-of-function mutation in ZMPSTE24 [c.28_29insA, p.(Leu10Tyrfs*37)] in two consanguineous Pakistani families segregating MADB. To clarify how lethal consequences are prevented in affected individuals, functional analysis was performed. Expression experiments supported utilization of two alternative translation initiation sites, preventing complete loss of protein function consistent with the relatively mild phenotypic outcome in affected patients. One of these alternative start codons is newly formed at the insertion site. Our findings indicate that the creation of new potential start codons through N-terminal mutations in other disease-associated genes should generally be taken into consideration in the variant interpretation process.     

Internal initiation of translation of bovine viral diarrhea virus RNA.

Initiation of translation on the bovine viral diarrhea virus (BVDV) internal ribosomal entry site (IRES) was reconstituted in vitro from purified translation components to the stage of 48S ribosomal initiation complex formation. Ribosomal binding and positioning on this mRNA to form a 48S complex did not require the initiation factors eIF4A, eIF4B, or eIF4F, and translation of this mRNA was resistant to inhibition by a trans-dominant eIF4A mutant that inhibited cap-mediated initiation of translation. The BVDV IRES contains elements that are bound independently by ribosomal 40S subunits and by eukaryotic initiation factor (eIF) 3, as well as determinants that mediate direct attachment of 43S ribosomal complexes to the initiation codon.     

Trapping of messenger RNA by Fragile X Mental Retardation protein into cytoplasmic granules induces translation repression

Absence of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein, is responsible for the Fragile X syndrome, the most common form of inherited mental retardation. FMRP is a cytoplasmic protein associated with mRNP complexes containing poly(A)+mRNA. As a step towards understanding FMRP function(s), we have established the immortal STEK Fmr1 KO cell line and showed by transfection assays with FMR1-expressing vectors that newly synthesized FMRP accumulates into cytoplasmic granules. These structures contain mRNAs and several other RNA-binding proteins. The formation of these cytoplasmic granules is dependent on determinants located in the RGG domain. We also provide evidence that FMRP acts as a translation repressor following co-transfection with reporter genes. The FMRP-containing mRNPs are dynamic structures that oscillate between polyribosomes and cytoplasmic granules reminiscent of the Stress Granules that contain repressed mRNAs. We speculate that, in neurons, FMRP plays a role as a mRNA repressor in incompetent mRNP granules that have to be translocated from the cell body to distal locations such as dendritic spines and synaptosomes.     

Biosynthesis of reovirus-specified polypeptides: effect of point mutation of the sequences flanking the 5'-proximal AUG initiator codons of the reovirus S1 and S4 genes on the efficiency of mRNA translation

The effect on translation of site-directed nucleotide substitutions around the 5'-proximal AUG initiation codon of the reovirus s1 mRNA specifying polypeptide sigma 1 and the reovirus s4 mRNA specifying polypeptide sigma 3 was examined. The efficiency of synthesis of the S1-encoded sigma 1 polypeptide and the S4-encoded sigma 3 polypeptide was analyzed in transfected simian COS cells. Mutant s1 mRNAs possessing either GCU AUG G or GCA AUG G sequences surrounding the 5'-proximal sigma 1 AUG were translated with an efficiency comparable to that of the wild-type s1 mRNA which possesses the flanking sequence CCU AUG G. Mutant s4 mRNAs possessing either CCU AUG G or CCA AUG G sequences surrounding the 5'-proximal sigma 3 AUG were translated with an efficiency comparable to that of wild-type s4 mRNA which possesses the flanking sequence GCA AUG G. The s4 mRNAs, both wild-type and mutant, were translated in vivo about five times more efficiently than the s1 mRNAs, both wild-type and mutant. These results suggest that nucleotide positions other than the -3, -2, -1, and +4 positions relative to the 5'-proximal initiator AUG, where the A is +1, play a dominant role in determining the efficiency of translation of these two reovirus mRNAs in vivo.     

Mechanism of translation initiation by Dicistroviridae IGR IRESs

The Dicistroviridae is a growing virus family characterized by a dicistronic genome, wherein each open reading frame (ORF) is translated from an independent internal ribosome entry site (IRES). The 5′ IRES that translates the first open reading frame (ORF1) is similar to the picornaviral IRESs. However the second IRES, referred to as the intergenic region (IGR) IRES, - translates ORF2 by and uses an unusual mechanism of initiating protein synthesis. It folds into a compact RNA structure that can bind directly to 40S ribosomal subunits and form 80S complexes to initiate translation in the absence of any initiation factors. Despite its unusual mechanism, the IGR IRES has proven to be an elegant model for elucidating initiation mechanisms employed by IRESs, as well as making it a powerful research tool with diverse applications.