RNA polymerase I-promoted HIS4 expression yields uncapped, polyadenylated mRNA that is unstable and inefficiently translated in Saccharomyces cerevisiae.

RNA transcribed by RNA polymerase (pol) II undergoes a number of covalent modifications before being exported to the cytoplasm as mature mRNA and subsequently translated. Two such modifications, capping at the 5′ end and polyadenylation at the 3′ end of mRNA, are believed to be limited to the RNA pol II transcriptional machinery. The addition of the unique cap structure to the 5′ end of all mature eukaryotic mRNAs is tightly coupled to RNA pol II transcription as the cap can be detected when the 5′ end of mRNA emerges from the pol II transcriptional machinery (22, 32, 49). It has been shown that the cap structure is important for RNA transport, pre-RNA splicing, and mRNA stability (16, 23, 30, 40). One of the best-understood functions of the cap structure is its role in translation initiation. According to the ribosomal scanning model (34), the eukaryotic initiation factor eIF-4F complex is required for the binding of the ribosomal preinitiation complex to mRNAs and for unwinding secondary structure in the 5′ leader region. This allows the preinitiation complex to scan for the first downstream AUG start codon in a 5′-to-3′ direction (for reviews, see references 25 and 54). The well-accepted ribosomal scanning model (cap-dependent initiation mechanism) accounts for most of eukaryotic translation initiation events. However, a number of mRNAs have been described to be translated by a cap-independent mechanism of translation. For example, upon poliovirus infection of mammalian cells, the eIF-4F initiation complex is rendered nonfunctional as a result of proteolytic cleavage of the p220 subunit. This results in the shutdown of cap-dependent protein synthesis in host cells and allows preferential translation of uncapped viral mRNAs, which occurs by a cap-independent mechanism (reviewed by Sonenberg [55]). Cap-independent translation initiation has also been described for cellular mRNAs (38, 45). Previous studies have used in vivo-expressed capless mRNAs in mammalian cells to investigate the relationship between the cap structure and the translation efficiency (19, 20). However, these studies differ in conclusion as to whether a cap is needed for translation in these cells. The poly(A) tail at the 3′ end of eukaryotic mRNAs is another distinct feature of eukaryotic mRNAs. The polyadenylation step takes place in the nuclei posttranscriptionally. In short, the AAUAAA- and G/U-rich elements at the 3′ end of mRNA signal transcription termination and specific cleavage followed by consecutive addition of adenosine residues (8, 39, 58). McCracken et al. (39) have reported that the carboxy-terminal domain (CTD) of the pol II large subunit is required for efficient cleavage at the poly(A) site in vivo and that the CTD might associate with CPSF (cleavage and polyadenylation specificity factors) and CstF (cleavage stimulation factors) but not poly(A) polymerase (39), suggesting that the polyadenylation machinery, in part, associates with the pol II transcriptional apparatus. The synthesized tail is typically homogeneous in length, ranging from 75 nucleotides (nt) (40) in yeast cells to 200 nt (3) in mammalian cells. In the yeast Saccharomyces cerevisiae a general mechanism for mRNA decay whereby mRNA is first deadenylated down to approximately 10 A residues, which triggers the decapping of mRNA and subsequent 5′-to-3′ degradation of the message, has been proposed (2, 40). The 5′-to-3′ exonuclease that appears responsible for the bulk of mRNA degradation is encoded by the XRN1 gene. In addition to degradation of mRNA, poly(A) tails have also been implicated to be involved in translation. For example, poly(A)-tailed mRNAs are translated more efficiently than their deadenylated counterparts in rabbit reticulocyte lysates (31). In addition, genetic studies of yeast have implicated a connection between the poly(A) tails and the poly(A) binding protein (PABP) with translation of mRNA. A mutation in the SPB4 gene encoding one of the 60S ribosomal proteins can suppress the lethality resulting from a deletion of the pab1 gene, which encodes the major PABP in budding yeast. This finding suggested that PABP might be involved in translation (50). PABP-poly(A) complex has also been shown to enhance translation efficiency in vitro by recruiting 40S subunits to mRNAs, and this enhancement was cap binding protein (eIF-4E) independent (56). Recent studies have established a functional relationship between the cap structure at the 5′ end of mRNA and the poly(A) tail of mRNA. The Pab1 protein can copurify and coimmunoprecipitate with the eIF-4γ subunit of the eIF-4F complex in yeast extracts (57), suggesting that the 3′ and 5′ ends of mRNA may be near each other when mRNA is actively translated. Thus, a complex interplay exists between the cap and the poly(A) tail in both mRNA stability and translation initiation (6, 56, 57). In this study, we put the yeast HIS4 gene under the transcriptional control of the pol I ribosomal DNA (rDNA) promoter/enhancer region (referred to as the rhis4 gene) and extensively characterized the transcription of this gene, the 5′ and 3′ processing of transcripts, and the translational expression of rhis4 mRNA in yeast. Our analysis shows that RNA pol I promotion of HIS4 results in two mRNA species; one is the same size as the HIS4 mRNA, and the other is 1.3 kb longer than the HIS4 mRNA and is bicistronic. Surprisingly, both mRNAs appear to be polyadenylated despite transcription by RNA pol I. An xrn1Δ strain or a genetically isolated suppressor of rhis4, rhis1 (rhs stands for rhis4 suppressor) which encodes a defective xrn1 gene, results in a 9- to 10-fold increase in the combined levels of rhis4 mRNAs. This observation suggests that the majority of the rhis4 mRNAs are transcribed by pol I and degraded rapidly due to the absence of a cap structure. Despite a large increase in the level of rhis4 mRNAs in an rhs1 (xrn1) mutant strain, the level of His4 protein remains low relative to a HIS4+ strain. Our data suggest that in yeast cells, polyadenylation of rhis4 mRNAs is able to occur independently of RNA pol II transcription. In addition, our results point to the cap as being an essential element for efficient translation initiation in yeast cells.