Brief Summary of research
1. Structure of the endogenous spliceosome.
Splicing of pre-mRNA is a fundamental process of human gene regulation that occurs in a ribonucleoprotein (RNP) complex - the spliceosome. Splicing and alternative splicing are major contributors to the diversity of the human proteome, and changes in splicing and alternative splicing are hallmarks of many human diseases including cancer. Detailed mechanistic insight of the splicing reaction was gained from the study of the spliceosome assembled on a single intron in vitro. However, as most pre-mRNAs are multi-intronic that undergo alternative splicing, the endogenous splicing machine requires additional elements to those of the in vitro machine, to account for all these diverse functions. I have taken a different approach and studied the endogenous spliceosome – the supraspliceosome, isolated from mammalian cells. Together with Prof. Joseph Sperling we discovered and characterized the endogenous spliceosome – the supraspliceosome from mammalian cells, and we were the first to publish EM images of the endogenous spliceosome (Spann et al PNAS 1989),
We have demonstrated that the supraspliceosome is active in splicing and in alternative splicing (Azubel et al 2006; Sebbag-Sznajder et al 2012), and that it harbors the five-spliceosomal U snRNPs (U1, U2, U4, U5 and U6 snRNPs) during all stages of splicing. Structural studies of the endogenous spliceosome are performed using cutting-edge EM methods such as cryo-electron tomography and cryo-EM single particle techniques. Structural studies of the supraspliceosome, by electron tomography, (Sperling et al JMB 1997) and Cryo-electron tomography (Medalia et al J Struct Biol 2002) revealed that the supraspliceosome is composed of four native spliceosomes, each resembling the in vitro assembled spliceosome, which are connected by the pre-mRNA. We further determined the structure of the native spliceosome, the substructure of the supraspliceosome, by cryo-EM single particle technique at 20-Å resolution (Azubel et al 2004 #1082). This resolution was the highest available for a functional splicing complex until the recent revolution in cryo-EM in 2015. This structural work on the native spliceosome has become textbook material (Gene Control, 5th Edition, D. Latchman Editor; Molecular Biology: Genes to Proteins, 3rd edition, by Burton Tropp, Jones and Bartlett Publishers; Principles of Computational Cell Biology, by V Helms, John Wiley and Sons 2008; Molecular Biology: Structure and Dynamics of Genomes and Proteomes, by J Zlatanova and K E van Holde, Garland Science, 2015), as well as the supraspliceosome model (Molecular Biology: Structure and Dynamics of Genomes and Proteomes, by J Zlatanova and K E van Holde, Garland Science, 2015). Our structural work of the supraspliceosome now aims at achieving high-resolution structure of the native spliceosome and the supraspliceosome, taking advantage of the recent revolution in cryo-EM. We also developed gold-nanoclusters that can be attached to nucleic acids or proteins as tools to localize elements within the endogenous spliceosome using EM and cryo-EM.
2. A quality control mechanism of alternative splicing within the supraspliceosome. In the studies of supraspliceosome function, we have discovered a quality control mechanism that affects alternative splicing and operates within the supraspliceosome. This mechanism, which is conserved in evolution, prevents the production of defective mRNAs. However, under stress conditions and in cancer this mechanism is abrogated and thousands of defective human mRNAs are produced. A first clue to deciphering this mechanism was the identification of initiator-tRNA as an essential component of this mechanism, independent of its role in translation. Current research in my lab continues the efforts to decipher additional components of this mechanism and their mode of function.
We have recently discovered a novel SOS factor and will use gold-nanoclusters and electron microscopy to localize it within the supraspliceosome.
3. Novel functions for small non-coding RNA within the supraspliceosome. Since many small noncoding RNAs (sncRNAs) are embedded in introns, we reasoned that their processing should likely occur in supraspliceosomes. We have shown a cross talk between the microprocessor and the supraspliceosome. Furthermore, using RNA-Seq of small RNA within the supraspliceosome, pre-miRNAs, microRNAs and defined fragments of pre-miRNA were identified within it. Part of these small RNA sequences are unique to the supraspliceosome, and not found in the collection of microRNA in the cell, suggesting novel functions. We have recently demonstrated a role for supraspliceosomal mir-7704 in negative regulating the expression of lincRNA HAGLR (Mahlab-Aviv et al NAR 2018). Another group of sncRNAs found within the supraspliceosome is C/D box snoRNAs (SNORDs), whose known role is in methylating ncRNA via their methylase fibrillarin. Importantly, in collaboration with the Stamm lab (University of Lexington) we identified a subgroup of SNORDs within the supraspliceosome. Notably, this group lacks their canonical methylating enzyme fibrillarin, suggesting a novel function. We demonstrated that this class of SNORDs have a role in regulating alternative splicing. Current work in my lab on the supraspliceosomal miRNAs focuses on changes in supraspliceosome miRNAs in cancer, revealing changes in their expression during the development of the disease. Identification of the targets of these sncRNAs and their effect on gene expression might lead to new avenues in the struggle against cancer. Work on supraspliceosomal SNORDs aims at characterizing the structure of supraspliceosomal SNORDs and deciphering their function.
4. The supraspliceosome – the endogenous pre-mRNA processing machine. The combined studies of the above three aims should enable us to decipher the mode of action of the supraspliceosome as the pre-mRNA processing machine. The supraspliceosome is a stand-alone macromolecular machine, composed of four native spliceosomes that are connected by a single pre-mRNA molecule, capable of performing splicing of its pre-mRNA. Importantly, different transcripts independent on their length or number of introns are found individually assembled in a supraspliceosome, indicating the universal nature of the supraspliceosome. The supraspliceosome enable communication between the native spliceosomes, which is a crucial element for regulated alternative splicing and for quality control of the resulting mRNAs. The supraspliceosome structure provides a platform to juxtapose exons about to be spliced, and each of the four native spliceosomes, can splice the intron wound around it. This multiprocessor machine can simultaneously splice four introns – not necessarily in a consecutive manner. This configuration enables examination, prior to introns excision, if correct splice junctions will be combined, and allows rearrangement of splice junction combinations to select the appropriate ones, thus ensuring the fidelity of splicing and alternative splicing. The recent revolution in cryo-EM brought high resolution structures of spliceosome intermediates, revealing important information about dynamic interactions within the spliceosome during the splicing reaction. Yet, the only available structure that can account for interactions between spliceosomes in alternative splicing is the supraspliceosome. The supraspliceosome model predicts that each transcript will be assembled in a tetrameric supraspliceosome. Splicing of a multi-intronic pre-mRNA can be facilitated by the translocation of the pre-mRNA through the complex in a ‘rolling model’ fashion. After processing of four introns the RNA roles in to place a new subset of introns for processing. Transcripts with less than four introns are also assembled in supraspliceosomes, the interactions of the RNA with the native spliceosomes are presumably sufficient to hold the structure together. It is established, that unlike the in-vitro spliceosome, all five-spliceosomal U snRNPs are associated with the supraspliceosome at all stages of splicing. The supraspliceosome harbors components of all pre-mRNA processing activities, thus representing the nuclear pre-mRNA processing machine, capable of performing all the pre-mRNA processing activities that the pre-mRNA has to undergo before it can exit from the nucleus to the cytoplasm. The research in the above three topic will allow us to confirm these hypothesis and obtained the detailed dynamics of this important and complex machine