Juan Valcárcel

Alternative pre-mRNA splicing: the logic of combinatorial control

Alternative splicing of mRNA precursors is a versatile mechanism of gene expression regulation that accounts for a considerable proportion of proteomic complexity in higher eukaryotes. Its modulation is achieved through the combinatorial interplay of positive and negative regulatory signals present in the RNA, which are recognized by complexes composed of members of the hnRNP and SR protein families.

Alternative splicing and genome complexity

Alternative splicing of mRNA allows many gene products with different functions to be produced from a single coding sequence. It has recently been proposed as a mechanism by which higher-order diversity is generated. Here we show, using large-scale expressed sequence tag (EST) analysis, that among seven different eukaryotes the amount of alternative splicing is comparable, with no large differences between humans and other animals.

Nucleosome positioning as a determinant of exon recognition

Chromatin structure influences transcription, but its role in subsequent RNA processing is unclear. Here we present analyses of high-throughput data that imply a relationship between nucleosome positioning and exon definition. First, we have found stable nucleosome occupancy within human and Caenorhabditis elegans exons that is stronger in exons with weak splice sites. Conversely, we have found that pseudoexons—intronic sequences that are not included in mRNAs but are flanked by strong splice sites—show nucleosome depletion. Second, the ratio between nucleosome occupancy within and upstream from the exons correlates with exon-inclusion levels. Third, nucleosomes are positioned central to exons rather than proximal to splice sites. These exonic nucleosomal patterns are also observed in non-expressed genes, suggesting that nucleosome marking of exons exists in the absence of transcription. Our analysis provides a framework that contributes to the understanding of splicing on the basis of chromatin architecture.

The SR protein family: pleiotropic functions in pre-mRNA splicing

A family of proteins with arginine-serine-rich domains has recently come into the limelight of studies on the mechanisms of constitutive and regulated pre-mRNA splicing. Implicated in an ever increasing variety of functions, these proteins act as driving forces during spliceosome assembly and also play decisive roles in alternative splice-site selection, suggesting that they are crucial players in the regulation of splicing during cell differentiation and development.

Inhibition of msl-2 splicing by Sex-lethal reveals interaction between U2AF35 and the 3' splice site AG.

The protein Sex-lethal (SXL) controls dosage compensation in Drosophila by inhibiting the splicing and translation of male-specific-lethal-2 (msl-2) transcripts. Here we report that splicing inhibition of msl-2 requires a binding site for SXL at the polypyrimidine (poly(Y)) tract associated with the 3′ splice site, and an unusually long distance between the poly(Y) tract and the conserved AG dinucleotide at the 3′ end of the intron. Only this combination allows efficient blockage of U2 small nuclear ribonucleoprotein particle binding and displacement of the large subunit of the U2 auxiliary factor (U2AF65) from the poly(Y) tract by SXL. Crosslinking experiments with ultraviolet light indicate that the small subunit of U2AF (U2AF35) contacts the AG dinucleotide only when located in proximity to the poly(Y) tract. This interaction stabilizes U2AF65 binding such that SXL can no longer displace it from the poly(Y) tract. Our results reveal a novel function for U2AF35, a critical role for the 3′ splice site AG at the earliest steps of spliceosome assembly and the need for a weakened U2AF35–AG interaction to regulate intron removal.

The spliceosome as a target of novel antitumour drugs

Several bacterial fermentation products and their synthetic derivatives display antitumour activities and bind tightly to components of the spliceosome, which is the complex molecular machinery involved in the removal of introns from mRNA precursors in eukaryotic cells. The drugs alter gene expression, including alternative splicing, of genes that are important for cancer progression. A flurry of recent reports has revealed that genes encoding splicing factors, including the drug target splicing factor 3B subunit 1 (SF3B1), are among the most highly mutated in various haematological malignancies such as chronic lymphocytic leukaemia and myelodysplastic syndromes. These observations highlight the role of splicing factors in cancer and suggest that an understanding of the molecular effects of drugs targeting these proteins could open new perspectives for studies of the spliceosome and its role in cancer progression, and for the development of novel antitumour therapies.

Building specificity with nonspecific RNA-binding proteins

Specificity is key to biological regulation. Two families of RNA binding proteins, heterogeneous nuclear ribonucleoproteins and serine-arginine–rich proteins, were initially thought to have redundant or nonspecific biochemical functions. Recently, members of these families have been found as components of distinct regulatory complexes with highly specific and essential roles in mRNA metabolism. Here we discuss the basis for their functional specificity and the mechanisms of action of some of their characteristic protein domains.

The splicing regulator TIA-1 interacts with U1-C to promote U1 snRNP recruitment to 5' splice sites.

The U1 small nuclear ribonucleoprotein (U1 snRNP) binds to the pre‐mRNA 5′ splice site (ss) at early stages of spliceosome assembly. Recruitment of U1 to a class of weak 5′ ss is promoted by binding of the protein TIA‐1 to uridine‐rich sequences immediately downstream from the 5′ ss. Here we describe a molecular dissection of the activities of TIA‐1. RNA recognition motifs (RRMs) 2 and 3 are necessary and sufficient for binding to the pre‐mRNA. The non‐ consensus RRM1 and the C‐terminal glutamine‐rich (Q) domain are required for association with U1 snRNP and to facilitate its recruitment to 5′ ss. Co‐precipitation experiments revealed a specific and direct interaction involving the N‐terminal region of the U1 protein U1‐C and the Q‐rich domain of TIA‐1, an interaction enhanced by RRM1. The results argue that binding of TIA‐1 in the vicinity of a 5′ ss helps to stabilize U1 snRNP recruitment, at least in part, via a direct interaction with U1‐C, thus providing one molecular mechanism for the function of this splicing regulator.

The hnRNP A1 protein regulates HIV‐1 tat splicing via a novel intron silencer element

The generation of >30 different HIV‐1 mRNAs is achieved by alternative splicing of one primary transcript. The removal of the second tat intron is regulated by a combination of a suboptimal 3′ splice site and cis‐acting splicing enhancers and silencers. Here we show that hnRNP A1 inhibits splicing of this intron via a novel heterogeneous nuclear ribonucleoprotein (hnRNP) A1‐responsive intron splicing silencer (ISS) that can function independently of the previously characterized exon splicing silencer (ESS3). Surprisingly, depletion of hnRNP A1 from the nuclear extract (NE) enables splicing to proceed in NE that contains 100‐fold reduced concentrations of U2AF and normal levels of SR proteins, conditions that do not support processing of other efficiently spliced pre‐mRNAs. Reconstituting the extract with recombinant hnRNP A1 protein restores splicing inhibition at a step subsequent to U2AF binding, mainly at the time of U2 snRNP association. hnRNP A1 interacts specifically with the ISS sequence, which overlaps with one of three alternative branch point sequences, pointing to a model where the entry of U2 snRNP is physically blocked by hnRNP A1 binding.

Multi-domain conformational selection underlies pre-mRNA splicing regulation by U2AF

Many cellular functions involve multi-domain proteins, which are composed of structurally independent modules connected by flexible linkers. Although it is often well understood how a given domain recognizes a cognate oligonucleotide or peptide motif, the dynamic interaction of multiple domains in the recognition of these ligands remains to be characterized. Here we have studied the molecular mechanisms of the recognition of the 3′-splice-site-associated polypyrimidine tract RNA by the large subunit of the human U2 snRNP auxiliary factor (U2AF65) as a key early step in pre-mRNA splicing. We show that the tandem RNA recognition motif domains of U2AF65 adopt two remarkably distinct domain arrangements in the absence or presence of a strong (that is, high affinity) polypyrimidine tract. Recognition of sequence variations in the polypyrimidine tract RNA involves a population shift between these closed and open conformations. The equilibrium between the two conformations functions as a molecular rheostat that quantitatively correlates the natural variations in polypyrimidine tract nucleotide composition, length and functional strength to the efficiency to recruit U2 snRNP to the intron during spliceosome assembly. Mutations that shift the conformational equilibrium without directly affecting RNA binding modulate splicing activity accordingly. Similar mechanisms of cooperative multi-domain conformational selection may operate more generally in the recognition of degenerate nucleotide or amino acid motifs by multi-domain proteins.