Witold Filipowicz

Functions of the 5′-terminal m7G cap in eukaryotic mRNA

All cellular eukaryotic mRNAs studied so far contain at their 5’-terminus a blocked methylatcd structure often referred to as a ‘cap’. Most of the eukaryotic viral mRNAs are similarly modified. The general formula for the capped 5’-terminus of mRNAs may be represented as m7G(5’)ppp(5’)X(m)pY(mI. In this structure the 7-methylguanosine (m”G) and the penultimate nucleoside (X) are joined by their .5’-hydroxyl groups through a triphosphate bridge. Nucfeosides X and Y are often also methyfated in the 2’Oposition and an ~‘,2’~~imethyladenosine may be found in position X. The 26 S mRNA coded in vivo by Sindbis virus contains, in addition to the usual m7G, also mz2”G or m32s2J7G at its 5’-terminus PI f This article is concerned with the possible role played by the 5’-terminal cap in eukaryotic mRNA. For more details on the cap structure, its occurrence in different mRNA species, the mechanism of cap synthesis and earlier studies on its function, the reader is referred to the review by Shatkin [3] a Based on extensive in vitro translation studies two main functions have emerged for the .S’-terminal

A chloroplastic RNA-binding protein is a new member of the PPR family

P67, a new protein binding to a specific RNA probe, was purified from radish seedlings [Echeverria, M. and Lahmy, S. (1995) Nucleic Acids Res. 23, 4963–4970]. Amino acid sequence information obtained from P67 microsequencing allowed the isolation of genes encoding P67 in radish and Arabidopsis thaliana. Immunolocalisation experiments in transfected protoplasts demonstrated that this protein is addressed to the chloroplast. The RNA‐binding activity of recombinant P67 was found to be similar to that of the native protein. A significant similarity with the maize protein CRP1 [Fisk, D.G., Walker, M.B. and Barkan, A. (1999) EMBO J. 18, 2621–2630] suggests that P67 belongs to the PPR family and could be involved in chloroplast RNA processing.

Translation of potato virus x RNA into high molecular weight proteins

Potato virus X (PVX) is a flexous rod-shaped virus containing a single molecule of genomic RNA of 2 X lo6 mol. wt [I]. Recently some data on the structure and messenger activity of PVX RNA has become available: this RNA has a m7 GpppG cap at the S’-te~n~inus and its 3’-end is not polyadenylated (21. Furthermore PVX RNA is an active template for in vitro protein synthesis in wheat germ extract; the largest translation product coded by PVX RNA was found to have an app. mol. wt 110 000 [3]. We have used an mRNA-dependent reticulocyte lysate and a wheat germ extract to study the polypeptides coded by PVX RNA. In both systems PVX RNA was translated into two high molecular weight proteins of 180 000 and 145 000. Experimental evidence indicates that these two proteins share common amino acid sequences and that their synthesis may occur by a mechanism similar to that of TMV RNA or TYMV RNA-coded polypeptides.

Faithful and efficient translation of viral and cellular eukaryotic mRNAs in a cell-free S-27 extract of Saccharomycescerevisiae

Abstract The preparation of yeast spheroplast 27,000 × g supernatant /S-27/ which initiates translation of endogenous and exogenous mRNAs is described. The activity of this protein synthesis system is comparable to that of the most efficient cell-free extracts currently in use. The yeast S-27 system is able to carry out faithful translation of distinct eukaryotic mRNAs into proteins as large as 180,000 daltons.

RNA ligation in eukaryotes

Abstract Two distinct enzymatic RNA ligation pathways operate in plant and animal cell extracts. They both function in tRNA splicing and possibly in other RNA processing events. Ligation of two exons during Tetrahymena pre-rRNA splicing is coupled with the excision of introns and does not require any protein.

f2 RNA structure and peptide chain initiation: fMet-tRNA binding directed by methoxyamine-modified unfolded or native-like f2 RNAs

Abstract f2 RNA in which all exposed, unpaired cytosines are modified with methoxyamine retains an unchanged ordered structure. This RNA shows the same capacity to direct fMet-tRNA binding to E. coli ribosomes as unmodified f2 RNA. As this binding corresponds mainly to the coat protein initiation site, our results suggest that unpaired cytosines present in the coat protein initiation fragment are not involved in the specific recognition of that fragment by ribosomes. When f2 RNA is treated with methoxyamine in the presence of guanidine-HCl the resulting modified f2 RNA becomes irreversibly unfolded. Such RNA stimulates fMet-tRNA binding about 50 times more efficiently than native f2 RNA. E. coli ribosomal RNA when unfolded under the same conditions also stimulates very efficiently the binding of fMet-tRNA.

Reaction of methoxyamine with phage f2 RNA and activity of modified messenger

Mode of action of hydroxylamine and methoxyamine on cytosine ring is well established [l-4]. Methoxyamine, in contrast to hydroxylamine, is not reactive towards uracil [S] and therefore can be used as a selective reagent in studies on nucleic acid structure and function. Cashmore et al. [6] have recently shown that in the case of E. coli tRNA, methoxyamine reacts mainly with sterically exposed cytosines which represent only about 20% of total cytosine content. This work shows that methoxyamine can serve for structural and functional studies of natural messenger such as phage f2 RNA. The kinetics of reaction of methoxyamine with f2 RNA was studied using “C-labelled reagent. The results of melting and sedimentation analyses of methoxyamine-modified f2 RNA indicate that under conditions employed, methoxyamine treatment does not change the higher structure of f2 RNA molecules. However, modified f2 RNA alters significantly its capacity to code for phage specific polypeptides.