Our research currently focuses on three distinct but interconnected areas involving the basic mechanisms of eukaryotic gene expression: (1) the structure and mechanism of the spliceosome, (2) the effects of nuclear-acquired proteins on cytoplasmic messenger RNA (mRNA) metabolism, and (3) the fate of functionally defective ribosomal RNAs (rRNAs) and mRNAs.
Structural and Mechanistic Studies of Spliceosomes
Introns are incoherent strings of nucleotides that interrupt the coding regions of genes. They are removed from nascent RNA transcripts by the process of precursor mRNA (pre-mRNA) splicing. Since the majority of genes in multicellular organisms contain introns, their timely and precise removal is essential for proper gene expression. Most introns are excised by the major spliceosome, a complex macromolecular machine containing five stable small nuclear RNAs (snRNAs) and scores of proteins. The spliceosome must be at once precise (e.g., a 1-nucleotide shift in a splice site will throw the protein-coding region completely out of frame) and adaptable (in humans it must recognize >105 different splice site pairs in diverse sequence contexts). In metazoans, the recognition problem is compounded by poor conservation of the sequences defining splice sites and the presence of multiple introns per pre-mRNA. Also, a remarkably high percentage of metazoan pre-mRNAs are subject to alternative splicing, which greatly expands the repertoire of proteins that can be expressed from relatively small genomes. Thus the splicing machinery must be highly malleable to allow for regulated alternate splice site choice.
A central goal of our research is to elucidate the basic mechanisms by which spliceosomes accurately identify splice sites in pre-mRNAs and then catalyze intron excision. A current emphasis in our lab is the development of methodologies for following reactions to pre-mRNA splicing in real time, using multiwavelength single-molecule fluorescence (SMF). All previous in vitro mechanistic studies of splicing have used bulk assays that report only the average behavior of a population. Although such ensemble assays have provided a wealth of mechanistic insight, they are limited in their ability to tease out finer mechanistic details. Single-molecule methods, on the other hand, permit observation of the stochastic behavior of individual binding and catalytic events, allowing analysis of many events that would otherwise go undetected.
Using a pre-mRNA attached to a glass surface via its 3' end and containing fluorescent labels in the 5' exon and intron, we are now able to observe individual splicing events in Saccharomyces cerevisiae extracts, using a multiwavelength total internal reflection fluorescence (TIRF) microscope system developed by Jeff Gelles (Brandeis University) and his colleagues. We are also using a diverse array of chemical biology tools to fluorescently label core spliceosomal proteins as well as numerous transiently associating splicing factors. By allowing us to analyze the dynamic characteristics of individual spliceosomes in real time, SMF has opened an exciting new window onto previously unaddressable questions regarding spliceosome assembly and internal structural transitions, as well as the comings and goings of key splicing factors.
Effects of Nuclear-Acquired Proteins on Cytoplasmic mRNA Metabolism
In addition to removing introns, the process of pre-mRNA splicing has significant consequences for subsequent metabolism of the product mRNA. That is, mRNAs produced by splicing are subject to different subcellular localization, different efficiencies of translation into proteins, and different decay rates than otherwise identical mRNAs produced from intronless genes. Splicing affects downstream mRNA metabolism by altering the complement of proteins associated with the mRNP (mRNA ribonucleoprotein particle). Such nuclear-acquired proteins accompany the spliced mRNP to the cytoplasm, where they interface with a variety of machineries and signaling pathways to fine tune gene expression.
One set of nuclear-acquired proteins is the exon junction complex (EJC), which is stably deposited by the spliceosome 20–24 nucleotides upstream of mRNA exon-exon junctions. Although the EJC is loaded at a specific position, its binding is virtually independent of RNA sequence. We identified the species responsible for this stable but sequence-independent binding as the DEAD-box protein eIF4AIII. Because of their strong homology to the SF2 family of DNA helicases, DEAD-box proteins are often presumed to function as RNA helicases or RNPases. However, our finding that eIF4AIII acts as a stable, sequence-independent RNA binder suggests such proteins can also act as RNA "placeholders" or "clothespins" rather than RNA translocases. Such molecular clothespins could serve as a general means for attaching factors that add functionality to an RNP as a consequence of some process to which the RNA has been exposed (e.g., splicing), without requiring any special consensus sequences in the RNA. We are exploring the idea that other DEAD-box proteins act as molecular clothespins to facilitate a variety of processes involving RNA.
Another area of active investigation concerns the effects of nuclear-acquired EJC components on mRNA utilization and metabolism. In collaboration with Gina Turrigiano (Brandeis University) and Chris Burge (Massachusetts Institute of Technology), we recently showed that eIF4AIII remains associated with dendritically localized mRNAs in mammalian neurons and acts as a key brake on expression of proteins required for synaptic function. One mechanism for this braking action is via the translation-dependent decay of Arc mRNA, the gene for which contains two conserved introns in its 3'-untranslated region (3'-UTR). This is a highly unusual gene structure in mammals, as EJCs downstream of stop codons trigger nonsense-mediated mRNA decay (NMD). A bioinformatics approach revealed 148 other mammalian genes with this same feature, suggesting that translation-dependent mRNA decay mechanisms such as NMD might be widely employed in mammalian cells as a means to limit the amount of protein produced from certain mRNAs. Curiously, a large number of these genes are expressed in hematopoietic cells, suggesting that some feature of blood cells may particularly favor their evolution there. Future experiments will probe the role of the EJC in modulating expression from some of these new 3'-UTR intron-containing genes.
Clearance of Nonfunctional RNAs
Eukaryotes possess numerous quality control systems that monitor both the synthesis of RNA and the integrity of the finished products. Over the past two decades, numerous pathways have been described for mRNA quality control. All of these pathways are translation-dependent, initiating when a ribosome stalls during translation in a context that impedes efficient elongation or termination. Nonstop mRNA decay (NSD) eliminates mRNAs lacking any in-frame stop codon, such as truncated or prematurely polyadenylated transcripts. NMD eliminates mRNAs containing a stop codon in a poor context for translation termination, often a nonsense or premature termination codon. Finally, no-go mRNA decay (NGD) eliminates mRNAs containing a structural barrier within the open reading frame that induces ribosome stalling.
In addition to these mRNA-specific pathways, we recently demonstrated that S. cerevisiae possess a novel quality control mechanism, nonfunctional rRNA decay (NRD), capable of detecting and eliminating translationally defective rRNAs. Our most recent data indicate that NRD can be divided into two separate pathways, one specific for the 25S rRNA present in the large ribosomal subunit (25S NRD), and a second specific for the 18S rRNA present in the small ribosomal subunit (18S NRD). Notably, 18S NRD shares many similarities (e.g., trans-acting factor requirements, subcellular localization) with NGD, suggesting that 18S NRD and NGD are different observable outcomes of the same initiating event: a ribosome stalled inappropriately at a sense codon during translation elongation. We are investigating the possibility that such ribosome stalling can be caused by damaged nucleotides in either the mRNA or rRNA. Thus one function of the 18S NRD/NGD pathway may be to clear the cell of damaged RNAs. Significantly, oxidatively damaged RNAs have been implicated in neurodegenerative diseases, whereas alkylated RNAs are by-products of many alkylating anticancer regimens.
