Deciphering 3'ss selection in the yeast genome reveals an RNA thermosensor that mediates alternative splicing.

Poor understanding of the spliceosomal mechanisms to select intronic 3' ends (3'ss) is a major obstacle to deciphering eukaryotic genomes. Here, we discern the rules for global 3'ss selection in yeast. We show that, in contrast to the uniformity of yeast splicing, the spliceosome uses all available 3'ss within a distance window from the intronic branch site (BS), and that in ∼70% of all possible 3'ss this is likely to be mediated by pre-mRNA structures. Our results reveal that one of these RNA folds acts as an RNA thermosensor, modulating alternative splicing in response to heat shock by controlling alternate 3'ss availability. Thus, our data point to a deeper role for the pre-mRNA in the control of its own fate, and to a simple mechanism for some alternative splicing.

DNA structure directs positioning of the mitochondrial genome packaging protein Abf2p.

The mitochondrial genome (mtDNA) is assembled into nucleo-protein structures termed nucleoids and maintained differently compared to nuclear DNA, the involved molecular basis remaining poorly understood. In yeast (Saccharomyces cerevisiae), mtDNA is a ∼80 kbp linear molecule and Abf2p, a double HMG-box protein, packages and maintains it. The protein binds DNA in a non-sequence-specific manner, but displays a distinct 'phased-binding' at specific DNA sequences containing poly-adenine tracts (A-tracts). We present here two crystal structures of Abf2p in complex with mtDNA-derived fragments bearing A-tracts. Each HMG-box of Abf2p induces a 90° bend in the contacted DNA, causing an overall U-turn. Together with previous data, this suggests that U-turn formation is the universal mechanism underlying mtDNA compaction induced by HMG-box proteins. Combining this structural information with mutational, biophysical and computational analyses, we reveal a unique DNA binding mechanism for Abf2p where a characteristic N-terminal flag and helix are crucial for mtDNA maintenance. Additionally, we provide the molecular basis for A-tract mediated exclusion of Abf2p binding. Due to high prevalence of A-tracts in yeast mtDNA, this has critical relevance for nucleoid architecture. Therefore, an unprecedented A-tract mediated protein positioning mechanism regulates DNA packaging proteins in the mitochondria, and in combination with DNA-bending and U-turn formation, governs mtDNA compaction.

L30 binds the nascent RPL30 transcript to repress U2 snRNP recruitment.

The mechanisms of pre-mRNA splicing regulation are poorly understood. Here we dissect how the Saccharomyces cerevisiae ribosomal L30 protein blocks splicing of its pre-mRNA upon binding a kink-turn structure including the 5' splice site. We show that L30 binds the nascent RPL30 transcript without preventing recognition of the 5' splice site by U1 snRNP but blocking U2 snRNP association with the branch site. Interaction of the factors BBP and Mud2 with the intron, relevant for U2 snRNP recruitment, is not affected by L30. Furthermore, the functions of neither the DEAD-box protein Sub2 in the incipient spliceosome nor the U2 snRNP factor Cus2 on branch site recognition are required for L30 inhibition. These findings contrast with the effects caused by binding a heterologous protein to the same region, completely blocking intron recognition. Collectively, our data suggest that L30 represses a spliceosomal rearrangement required for U2 snRNP association with the transcript.

RNA secondary structure mediates alternative 3'ss selection in Saccharomyces cerevisiae.

Alternative splicing is the mechanism by which different combinations of exons in the pre-mRNA give rise to distinct mature mRNAs. This process is mediated by splicing factors that bind the pre-mRNA and affect the recognition of its splicing signals. Saccharomyces species lack many of the regulatory factors present in metazoans. Accordingly, it is generally assumed that the amount of alternative splicing is limited. However, there is recent compelling evidence that yeast have functional alternative splicing, mainly in response to environmental conditions. We have previously shown that sequence and structure properties of the pre-mRNA could explain the selection of 3' splice sites (ss) in Saccharomyces cerevisiae. In this work, we extend our previous observations to build a computational classifier that explains most of the annotated 3'ss in the CDS and 5' UTR of this organism. Moreover, we show that the same rules can explain the selection of alternative 3'ss. Experimental validation of a number of predicted alternative 3'ss shows that their usage is low compared to annotated 3'ss. The majority of these alternative 3'ss introduce premature termination codons (PTCs), suggesting a role in expression regulation. Furthermore, a genome-wide analysis of the effect of temperature, followed by experimental validation, yields only a small number of changes, indicating that this type of regulation is not widespread. Our results are consistent with the presence of alternative 3'ss selection in yeast mediated by the pre-mRNA structure, which can be responsive to external cues, like temperature, and is possibly related to the control of gene expression.

Regulated pre-mRNA splicing: the ghostwriter of the eukaryotic genome.

Intron removal is at the heart of mRNA synthesis. It is mediated by one of the cell's largest complexes, the spliceosome. Yet, the fundamental chemistry involved is simple. In this review we will address how the spliceosome acts in diverse ways to optimize gene expression in order to meet the cell's needs. This is done largely by regulating the splicing of key transcripts encoding products that control gene expression pathways. This widespread role is evident even in the yeast Saccharomyces cerevisiae, where many introns appear to have been lost; yet how this control is being achieved is known only in a few cases. Here we explore the relevant examples and posit hypotheses whereby regulated splicing fine-tunes gene expression pathways to maintain cell homeostasis. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.

SUS1 introns are required for efficient mRNA nuclear export in yeast.

Efficient coupling between mRNA synthesis and export is essential for gene expression. Sus1/ENY2, a component of the SAGA and TREX-2 complexes, is involved in both transcription and mRNA export. While most yeast genes lack introns, we previously reported that yeast SUS1 bears two. Here we show that this feature is evolutionarily conserved and critical for Sus1 function. We determine that while SUS1 splicing is inefficient, it responds to cellular conditions, and intronic mutations either promoting or blocking splicing lead to defects in mRNA export and cell growth. Consistent with this, we find that an intron-less SUS1 only partially rescues sus1Δ phenotypes. Remarkably, splicing of each SUS1 intron is also affected by the presence of the other and by SUS1 exonic sequences. Moreover, by following SUS1 RNA and protein levels we establish that nonsense-mediated decay (NMD) pathway and the splicing factor Mud2 both play a role in SUS1 expression. Our data (and those of the accompanying work by Hossain et al.) provide evidence of the involvement of splicing, translation, and decay in the regulation of early events in mRNP biogenesis; and imply the additional requirement for a balance in splicing isoforms from a single gene.

RPL30 regulation of splicing reveals distinct roles for Cbp80 in U1 and U2 snRNP cotranscriptional recruitment.

Pre-mRNA splicing is catalyzed by the spliceosome, and its control is essential for correct gene expression. While splicing repressors typically interfere with transcript recognition by spliceosomal components, the yeast protein L30 blocks spliceosomal rearrangements required for the engagement of U2 snRNP (small ribonucleoprotein particle) to its own transcript RPL30. Using a mutation in the RPL30 binding site that disrupts this repression, we have taken a genetic approach to reveal that regulation of splicing is restored in this mutant by deletion of the cap-binding complex (CBC) component Cbp80. Indeed, our data indicate that Cbp80 plays distinct roles in the recognition of the intron by U1 and U2 snRNP. It promotes the initial 5' splice site recognition by U1 and, independently, facilitates U2 recruitment, depending on sequences located in the vicinity of the 5' splice site. These results reveal a novel function for CBC in splicing and imply that these molecular events can be the target of a splicing regulator.

The quest for a message: budding yeast, a model organism to study the control of pre-mRNA splicing.

Removal of introns during pre-mRNA splicing is a critical process in gene expression, and understanding its control at both single-gene and genomic levels is one of the great challenges in Biology. Splicing takes place in a dynamic, large ribonucleoprotein complex known as the spliceosome. Combining Genetics and Biochemistry, Saccharomyces cerevisiae provides insights into its mechanisms, including its regulation by RNA-protein interactions. Recent genome-wide analyses indicate that regulated splicing is broad and biologically relevant even in organisms with a relatively simple intronic structure, such as yeast. Furthermore, the possibility of coordination in splicing regulation at genomic level is becoming clear in this model organism. This should provide a valuable system to approach the complex problem of the role of regulated splicing in genomic expression.

Intronic features that determine the selection of the 3' splice site.

Most eukaryotic primary transcripts include segments, or introns, that will be accurately removed during RNA biogenesis. This process, known as pre-messenger RNA splicing, is catalyzed by the spliceosome, accurately selecting a set of intronic marks from others apparently equivalent. This identification is critical, as incorrectly spliced RNAs can be toxic for the organism. One of these marks, the dinucleotide AG, signals the intronic 3' end, or 3' splice site (ss). In this review we will focus on those intronic features that have an impact on 3' ss selection. These include the location and type of neighboring sequences, and their distance to the 3' end. We will see that their interplay is needed to select the right intronic end, and that this can be modulated by additional intronic elements that contribute to alternative splicing, whereby diverse RNAs can be generated from identical precursors. This complexity, still poorly understood, is fundamental for the accuracy of gene expression. In addition, a clear knowledge of 3' ss selection is needed to fully decipher the coding potential of genomes.

Repositioning of the reaction intermediate within the catalytic center of the spliceosome.

Conformational change within the spliceosome is required between the first catalytic step of pre-mRNA splicing, when the branch site attacks the 5' splice site (SS), and the second step, when the 5' exon attacks the 3'SS. Little is known, however, about repositioning of the reaction substrates during this transition. Whereas the 5'SS is positioned for the first step by pairing with the invariant U6 snRNA-ACAGAG site, we demonstrate that this pairing interaction must be disrupted to allow transition to the second step. We propose that removal of the branch structure from the catalytic center is in competition with binding of the 3'SS substrate for the second step. Changes in the relative occupancy of first and second step substrates at the catalytic center alter efficiency of the two steps of splicing, allowing use of suboptimal intron sequences and thereby altering substrate selectivity.

Pre-spliceosome formation in S.pombe requires a stable complex of SF1-U2AF(59)-U2AF(23).

We have initiated a biochemical analysis of splicing complexes in extracts from the fission yeast Schizosaccharomyces pombe. Extracts of S.pombe contain high levels of the spliceosome-like U2/5/6 tri-snRNP, which dissociates into mono-snRNPs in the presence of ATP, and supports binding of U2 snRNP to the 3' end of introns, yielding a weak ATP-independent E complex and the stable ATP-dependent complex A. The requirements for S.pombe complex A formation (pre-mRNA sequence elements, protein splicing factors, SF1/BBP and both subunits of U2AF) are analogous to those of mammalian complex A. The S.pombe SF1/BBP, U2AF(59) and U2AF(23) are tightly associated in a novel complex that is required for complex A formation. This pre-formed SF1- U2AF(59)-U2AF(23) complex may represent a streamlined mechanism for recognition of the branch site, pyrimidine tract and 3' splice site at the 3' end of introns.