In budding yeast, the most abundantly spliced pre-mRNAs encode ribosomal proteins (RPs). To investigate the contribution of splicing to ribosome production and function, we systematically eliminated introns from all RP genes to evaluate their impact on RNA expression, pre-rRNA processing, cell growth, and response to stress. The majority of introns were required for optimal cell fitness or growth under stress. Most introns are found in duplicated RP genes, and surprisingly, in the majority of cases, deleting the intron from one gene copy affected the expression of the other in a nonreciprocal manner. Consistently, 70% of all duplicated genes were asymmetrically expressed, and both introns and gene deletions displayed copy-specific phenotypic effects. Together, our results indicate that splicing in yeast RP genes mediates intergene regulation and implicate the expression ratio of duplicated RP genes in modulating ribosome function.

Recent functional, proteomic and ribosome profiling studies in eukaryotes have concurrently demonstrated the translation of alternative open-reading frames (altORFs) in addition to annotated protein coding sequences (CDSs). We show that a large number of small proteins could in fact be coded by these altORFs. The putative alternative proteins translated from altORFs have orthologs in many species and contain functional domains. Evolutionary analyses indicate that altORFs often show more extreme conservation patterns than their CDSs. Thousands of alternative proteins are detected in proteomic datasets by reanalysis using a database containing predicted alternative proteins. This is illustrated with specific examples, including altMiD51, a 70 amino acid mitochondrial fission-promoting protein encoded in MiD51/Mief1/SMCR7L, a gene encoding an annotated protein promoting mitochondrial fission. Our results suggest that many genes are multicoding genes and code for a large protein and one or several small proteins.

In Saccharomyces cerevisiae, the maturation of both pre-rRNA and pre-small nucleolar RNAs (pre-snoRNAs) involves common factors, thereby providing a potential mechanism for the coregulation of snoRNA and rRNA synthesis. In this study, we examined the global impact of the double-stranded-RNA-specific RNase Rnt1p, which is required for pre-rRNA processing, on the maturation of all known snoRNAs. In silico searches for Rnt1p cleavage signals, and genome-wide analysis of the Rnt1p-dependent expression profile, identified seven new Rnt1p substrates. Interestingly, two of the newly identified Rnt1p-dependent snoRNAs, snR39 and snR59, are located in the introns of the ribosomal protein genes RPL7A and RPL7B. In vitro and in vivo experiments indicated that snR39 is normally processed from the lariat of RPL7A, suggesting that the expressions of RPL7A and snR39 are linked. In contrast, snR59 is produced by a direct cleavage of the RPL7B pre-mRNA, indicating that a single pre-mRNA transcript cannot be spliced to produce a mature RPL7B mRNA and processed by Rnt1p to produce a mature snR59 simultaneously. The results presented here reveal a new role of yeast RNase III in the processing of intron-encoded snoRNAs that permits independent regulation of the host mRNA and its associated snoRNA.Bacterial pre-rRNA processing is carried out by a defined set of nucleases (3-5, 43, 52). Key among this set is RNase III, initially isolated by its ability to bind and cleave duplex RNA (47, 48). RNase III generates the immediate precursors to the mature 16S and 23S rRNAs from the primary transcripts by cleaving within two extended RNA duplexes formed by longrange interactions that pair the termini of each rRNA (7, 63). These long-range interactions provide a simple method of coordinating the processing events at both ends of the transcript. In eukaryotes, pre-rRNA processing is more complex and requires many more small nucleolar RNAs (snoRNAs) and protein components with overlapping functions (13,15,16,41,46). For example, the removal of the 5Ј external-transcribed spacer requires 4 snoRNAs (U3, snR30, U14, and snR10) and about 64 snoRNAs are required for rRNA modifications (24, 57). snoRNAs are divided into two major subclasses: the first includes box C/D snoRNAs that function mostly as a guide for the methylation of rRNA (6, 20, 21, 55), while the second includes H/ACA snoRNAs that guide RNA pseudouridine formation (25,39,59). Most mammalian snoRNAs are encoded within intron sequences and are processed from either unspliced precursors or lariat species (18,19,64). In Saccharomyces cerevisiae, most snoRNAs are transcribed either as independent units or as a part of polycistronic transcript, while only 7 of the 66 known snoRNAs are located in the introns of mRNAs (14,44,53). Several polycistronic snoRNAs, and a few monocistronic ones, are processed by Rnt1p, the orthologue of the bacterial RNase III (30), which is also required for the processing of the pre-rRNA's 3Ј end (2,10,11,23,33). Following processing by Rnt1p, the RNAs are trimmed...

Transcription termination of messenger RNA (mRNA) is normally achieved by polyadenylation followed by Rat1p-dependent 5'-3' exoribonuleolytic degradation of the downstream transcript. Here we show that the yeast ortholog of the dsRNA-specific ribonuclease III (Rnt1p) may trigger Rat1p-dependent termination of RNA transcripts that fail to terminate near polyadenylation signals. Rnt1p cleavage sites were found downstream of several genes, and the deletion of RNT1 resulted in transcription readthrough. Inactivation of Rat1p impaired Rnt1p-dependent termination and resulted in the accumulation of 3' end cleavage products. These results support a model for transcription termination in which cotranscriptional cleavage by Rnt1p provides access for exoribonucleases in the absence of polyadenylation signals.

In baker's yeast, the majority of ribosomal protein genes (RPGs) are duplicated, and it was recently proposed that such duplications are preserved via the functional specialization of the duplicated genes. However, the origin and nature of duplicated RPGs' (dRPGs) functional specificity remain unclear. In this study, we show that differences in dRPG functions are generated by variations in the modality of gene expression and, to a lesser extent, by protein sequence. Analysis of the sequence and expression patterns of non-intron-containing RPGs indicates that each dRPG is controlled by specific regulatory sequences modulating its expression levels in response to changing growth conditions. Homogenization of dRPG sequences reduces cell tolerance to growth under stress without changing the number of expressed genes. Together, the data reveal a model where duplicated genes provide a means for modulating the expression of ribosomal proteins in response to stress.