Cap-dependent and cap-independent translation by internal initiation of mRNAs in cell extracts prepared from Saccharomyces cerevisiae.

Abstract

Translation extracts were prepared from various strains of Saccharomyces cerevisiae. The translation of mRNA molecules in these extracts was cooperatively enhanced by the presence of 5'-terminal cap structures and 3'-terminal poly(A) sequences. These cooperative effects could not be observed in other translation systems such as those prepared from rabbit reticulocytes, wheat germ, and human HeLa cells. Because the yeast translation system mimicked the effects of the cap structure and poly(A) tail on translational efficiency seen in vivo, this system was used to study cap-dependent and cap-independent translation of viral and cellular mRNA molecules. Both the 5' noncoding regions of hepatitis C virus and those of coxsackievirus Bi conferred cap-independent translation to a reporter coding region during translation in the yeast extracts; thus, the yeast translational apparatus is capable of initiating cap-independent translation. Although the translation of most yeast mRNAs was cap dependent, the unusually long 5' noncoding regions of mRNAs encoding cellular transcription factors TFIID and HAP4 were shown to mediate cap-independent translation in these extracts. Furthermore, both TFIID and HAP4 5' noncoding regions mediated translation of a second cistron when placed into the intercistronic spacer region of a dicistronic mRNA, indicating that these leader sequences can initiate translation by an internal ribosome binding mechanism in this in vitro translation system. This finding raises the possibility that an internal translation initiation mechanism exists in yeast cells for regulated translation of endogenous mRNAs.

Starting the protein synthesis machine: eukaryotic translation initiation

“…Later, a strong synergism between the cap structure and the poly(A) tail during translation initiation was discovered, first in electroporated cells (68) and subsequently in cell-free translation systems of different origins. (69)(70)(71)(72) In a yeast cell-free system, the poly(A) tail, like the cap structure, was able to support the recruitment of the 40S ribosomal subunit by itself to an uncapped mRNA. (73) This function of the poly(A) tail as well as the functional synergy with the cap structure requires the poly(A)-binding protein (PABP) (73) and its interaction with the N-terminal part of eIF4G (74,75) (Fig.…”

Specific RNP capture with antisense LNA/DNA mixmers

“…The Renilla luciferase version of these, i.e., p-Bluescript-BREF and p-Bluescript-BmR(EF)m, were generated by exchanging the firefly luciferase (Fluc) ORF by the Renilla version derived from the pRL-null plasmid (Promega), using XmaI and HpaI restriction sites. The plasmids bearing the Fluc ORF or chloramphenicol acetyl transferase (CAT) under the transcriptional control of T3 and T7 polymerases were previously described (Iizuka et al 1994;Gebauer et al 1999). Rluc, Fluc, and CAT reporter mRNAs were transcribed using the linearized (HindIII) plasmids in the presence of m7GpppG cap (KEDAR S.C.) using T7 or T3 in vitro transcription kits (MEGAscript; Thermo Fisher Scientific) following the manufacturer's recommendations.…”

Isolation and identification of short nucleotide sequences that affect translation initiation in Saccharomyces cerevisiae

“…These results suggest that cells can survive by using a cap-independent mechanism of initiating translation. Other studies have identified IRESes within the yeast TFIID and HAP4 mRNAs, which were shown to function in yeast cell-free lysates (21), and in the YAP1 and p150 mRNAs, which we showed can function in vegetatively growing cells (22). The ability of yeast cells to initiate translation internally is also supported by the observations that RNA sequences from various organisms and sources, including the cricket paralysis virus, can function as IRESes in yeast (23)(24)(25).…”