Splicing Intermediates Research Articles

Mammalian NET-seq analysis defines nascent RNA profiles and associated RNA processing genome-wide.

The transcription cycle of RNA polymerase II (Pol II) correlates with changes to the phosphorylation state of its large subunit C-terminal domain (CTD). We recently developed Native Elongation Transcript sequencing using mammalian cells (mNET-seq), which generates single-nucleotide-resolution genome-wide profiles of nascent RNA and co-transcriptional RNA processing that are associated with different CTD phosphorylation states. Here we provide a detailed protocol for mNET-seq. First, Pol II elongation complexes are isolated with specific phospho-CTD antibodies from chromatin solubilized by micrococcal nuclease digestion. Next, RNA derived from within the Pol II complex is size fractionated and Illumina sequenced. Using mNET-seq, we have previously shown that Pol II pauses at both ends of protein-coding genes but with different CTD phosphorylation patterns, and we have also detected phosphorylation at serine 5 (Ser5-P) CTD-specific splicing intermediates and Pol II accumulation over co-transcriptionally spliced exons. With moderate biochemical and bioinformatic skills, mNET-seq can be completed in ∼6 d, not including sequencing and data analysis.

Biogenesis, identification, and function of exonic circular RNAs.

Circular RNAs (circRNAs) arise during post‐transcriptional processes, in which a single‐stranded RNA molecule forms a circle through covalent binding. Previously, circRNA products were often regarded to be splicing intermediates, by‐products, or products of aberrant splicing. But recently, rapid advances in high‐throughput RNA sequencing (RNA‐seq) for global investigation of nonco‐linear (NCL) RNAs, which comprised sequence segments that are topologically inconsistent with the reference genome, leads to renewed interest in this type of NCL RNA (i.e., circRNA), especially exonic circRNAs (ecircRNAs). Although the biogenesis and function of ecircRNAs are mostly unknown, some ecircRNAs are abundant, highly expressed, or evolutionarily conserved. Some ecircRNAs have been shown to affect microRNA regulation, and probably play roles in regulating parental gene transcription, cell proliferation, and RNA‐binding proteins, indicating their functional potential for development as diagnostic tools. To date, thousands of ecircRNAs have been identified in multiple tissues/cell types from diverse species, through analyses of RNA‐seq data. However, the detection of ecircRNA candidates involves several major challenges, including discrimination between ecircRNAs and other types of NCL RNAs (e.g., trans‐spliced RNAs and genetic rearrangements); removal of sequencing errors, alignment errors, and in vitro artifacts; and the reconciliation of heterogeneous results arising from the use of different bioinformatics methods or sequencing data generated under different treatments. Such challenges may severely hamper the understanding of ecircRNAs. Herein, we review the biogenesis, identification, properties, and function of ecircRNAs, and discuss some unanswered questions regarding ecircRNAs. We also evaluate the accuracy (in terms of sensitivity and precision) of some well‐known circRNA‐detecting methods. WIREs RNA 2015, 6:563–579. doi: 10.1002/wrna.1294For further resources related to this article, please visit the WIREs website.

Circular RNA biogenesis can proceed through an exon-containing lariat precursor.

Pervasive expression of circular RNA is a recently discovered feature of eukaryotic gene expression programs, yet its function remains largely unknown. The presumed biogenesis of these RNAs involves a non-canonical 'backsplicing' event. Recent studies in mammalian cell culture posit that backsplicing is facilitated by inverted repeats flanking the circularized exon(s). Although such sequence elements are common in mammals, they are rare in lower eukaryotes, making current models insufficient to describe circularization. Through systematic splice site mutagenesis and the identification of splicing intermediates, we show that circular RNA in Schizosaccharomyces pombe is generated through an exon-containing lariat precursor. Furthermore, we have performed high-throughput and comprehensive mutagenesis of a circle-forming exon, which enabled us to discover a systematic effect of exon length on RNA circularization. Our results uncover a mechanism for circular RNA biogenesis that may account for circularization in genes that lack noticeable flanking intronic secondary structure.

Splicing of many human genes involves sites embedded within introns.

The conventional model for splicing involves excision of each intron in one piece; we demonstrate this inaccurately describes splicing in many human genes. First, after switching on transcription of SAMD4A, a gene with a 134 kb-long first intron, splicing joins the 3′ end of exon 1 to successive points within intron 1 well before the acceptor site at exon 2 is made. Second, genome-wide analysis shows that >60% of active genes yield products generated by such intermediate intron splicing. These products are present at ∼15% the levels of primary transcripts, are encoded by conserved sequences similar to those found at canonical acceptors, and marked by distinctive structural and epigenetic features. Finally, using targeted genome editing, we demonstrate that inhibiting the formation of these splicing intermediates affects efficient exon–exon splicing. These findings greatly expand the functional and regulatory complexity of the human transcriptome.

A homolog of lariat-debranching enzyme modulates turnover of branched RNA.

Turnover of the branched RNA intermediates and products of pre-mRNA splicing is mediated by the lariat-debranching enzyme Dbr1. We characterized a homolog of Dbr1 from Saccharomyces cerevisiae, Drn1/Ygr093w, that has a pseudo-metallophosphodiesterase domain with primary sequence homology to Dbr1 but lacks essential active site residues found in Dbr1. Whereas loss of Dbr1 results in lariat-introns failing broadly to turnover, loss of Drn1 causes low levels of lariat-intron accumulation. Conserved residues in the Drn1 C-terminal CwfJ domains, which are not present in Dbr1, are required for efficient intron turnover. Drn1 interacts with Dbr1, components of the Nineteen Complex, U2 snRNA, branched intermediates, and products of splicing. Drn1 enhances debranching catalyzed by Dbr1 in vitro, but does so without significantly improving the affinity of Dbr1 for branched RNA. Splicing carried out in in vitro extracts in the absence of Drn1 results in an accumulation of branched splicing intermediates and products released from the spliceosome, likely due to less active debranching, as well as the promiscuous release of cleaved 5'-exon. Drn1 enhances Dbr1-mediated turnover of lariat-intermediates and lariat-intron products, indicating that branched RNA turnover is regulated at multiple steps during splicing.

Analysis of Splicing In Vitro Using Extracts of Saccharomyces cerevisiae

In vitro splicing studies are a powerful means of investigating the requirements and mechanisms of action of the many components of the splicing apparatus. The ability to add and subtract components, purify activities, and reconstitute activity, as well as to expose the apparatus to chemical probes of various types, allows a far more mechanistically detailed view of the process to emerge than is available from genetic or in vivo studies alone. Two kinds of activities are assayed during in vitro splicing. The first concerns the chemical conversion of the substrate pre-mRNA into splicing intermediates and products and is usually visualized using a labeled substrate followed by separation on a denaturing gel. The second concerns the assembly of noncovalent complexes between the substrate and the myriad components of the splicing apparatus. This is also visualized using a labeled substrate, but the separation of complexes is achieved using native gel electrophoresis or gradient sedimentation. In this protocol, we describe the splicing reaction and its preparation for analysis by denaturing gels and native splicing complex gels. We also provide conditions for depletion of ATP, a critical cofactor that is hydrolyzed during numerous key steps in spliceosome assembly and splicing progression.

Biomimetic synthesis of cyclic peptides using novel thioester surrogates

Acyl shifts involving N-S and S-S rearrangements are reactions central to the breaking of a peptide bond and forming of thioester intermediates in an intein-catalyzed protein splicing that ultimately leads to the formation of a new peptide bond by an uncatalyzed S-N acyl shift reaction. To mimic these three acyl shift reactions in forming thioesters and the subsequent peptide ligation, here we describe the development of two 9-fluorenylmethoxycarbonyl (Fmoc)-compatible thioester surrogates that can undergo uncatalyzed N-S, S-S, and S-N acyl shifts for preparing thioesters and cyclic peptides. These surrogates were incorporated as a C-terminal amido moiety of a target peptide using Fmoc chemistry by solid-phase synthesis, and then transformed into a thioester or thiolactones via two acyl shift reactions with or without the presence of an external thiol under acidic conditions. The proposed intein-mimetic thioester surrogates were prepared using readily available starting materials including N-methyl cysteine or 2-thioethylbutylamide. A key functional moiety shared in their design is the thioethylamido (TEA) moiety, which is essential to effect a proximity-driven N-S acyl shift under a favorable five-member ring transition in the breaking of a peptide bond. Thus, the tandem series of acyl shifts effected by a TEA moiety in a thioester surrogate together with a thioethylamino moiety of an N-terminal Cys residue in a linear peptide precursor are chemical mimics of an intein, as they mediate both excision and ligation reactions in forming cyclic peptides including cyclic conotoxin and sunflower trypsin inhibitor described herein.

Abstract 2257: Thailanstatins: New pre-mRNA splicing inhibitors and potent antiproliferative agents discovered from Burkholderia Thailandensis MSMB43.

Abstract Natural products have been traditionally sought from actinomycetes, filamentous fungi and medicinal plants. Gram-negative bacterial species, such as Burkholderia, Chromobacterium, Lysobacter, Pseudomonas and Xenorhabdus, however, have recently captured attention as new sources of diverse bioactive natural products. Mining the genome sequence of B. thailandensis MSMB43 revealed a cryptic biosynthetic gene cluster highly resembling that of FR901,464, a prototype pre-mRNA splicing inhibitor produced by Pseudomonas sp. No. 2663. Transcriptioal analysis identified a cultivation condition in which a representive gene of the cryptic gene cluster is adequately expressed. Consequently, three new compounds, thailanstatins A, B and C, were isolated from the fermentation broth of B. thailandensis MSMB43 through natural product chemistry. Thailanstatins belong to the FR901,464-family of microbial products biosynthesized by a hybrid polyketide synthease-nonribosomal peptide synthetase pathway. They have an oveall structural similarity with FR901,464, but feature an extra acetic acid unit which endows the compouds with a significantly higher stability than FR901,464 under physiologically relevant conditions. In vitro assays showed that thailanstatins inhibit pre-mRNA splicing as potently as FR901,464, with half-maximal inhibitory concentrations in the single to sub μM range, causing pre-mRNA to accumulate and preventing the production of mRNA and splicing intermediates. In vitro cell culture assays indicated that thailanstatins also possess potent antiproliferative activities in representative human cancer cell lines, with half-maximal growth inhibitory concentrations in the single nM range (Table 1). This work provides new chemical entities for research and development, and validates the Burkholderia species as an exciting new source of bioactive natural products. Table 1.Antiproliferative activities of compounds in human cancer cell lines (GI50 in nM).CompoundDU-145 (prostate cancer)H232A (non-small cell lung cancer)MDA-MB235 (triple-negative breast cancer)SKOV-3 (ovarian cancer)Thailanstatin A1.11±0.022.26±0.172.58±0.112.69±0.37Thailanstatin B3.00±0.922.50±0.066.22±1.674.94±1.76Thailanstatin C2.98±0.903.67±0.538.82±2.205.57±2.01FR901,4641.05±0.021.94±0.242.10±0.191.06±0.01 Citation Format: Xiangyang Liu, Sreya Biswas, Michael G. Berg, Christopher M. Antapli, Feng Xie, Qi Wang, Man-Cheng Tang, Gong-Li Tang, Lixin Zhang, Gideon Dreyfuss, Yi-Qiang Cheng. Thailanstatins: New pre-mRNA splicing inhibitors and potent antiproliferative agents discovered from Burkholderia Thailandensis MSMB43. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2257. doi:10.1158/1538-7445.AM2013-2257

Intronic sequence elements impede exon ligation and trigger a discard pathway that yields functional telomerase RNA in fission yeast

The fission yeast telomerase RNA (TER1) precursor harbors an intron immediately downstream from its mature 3' end. Unlike most introns, which are removed from precursor RNAs by the spliceosome in two sequential but tightly coupled transesterification reactions, TER1 only undergoes the first cleavage reaction during telomerase RNA maturation. The mechanism underlying spliceosome-mediated 3' end processing has remained unclear. We now demonstrate that a strong branch site (BS), a long distance to the 3' splice site (3' SS), and a weak polypyrimidine (Py) tract act synergistically to attenuate the transition from the first to the second step of splicing. The observation that a strong BS antagonizes the second step of splicing in the context of TER1 suggests that the BS-U2 snRNA interaction is disrupted after the first step and thus much earlier than previously thought. The slow transition from first to second step triggers the Prp22 DExD/H-box helicase-dependent rejection of the cleaved products and Prp43-dependent "discard" of the splicing intermediates. Our findings explain how the spliceosome can function in 3' end processing and provide new insights into the mechanism of splicing.

Stalled Spliceosomes Are a Signal for RNAi-Mediated Genome Defense

Using the yeast Cryptococcus neoformans, we describe a mechanism by which transposons are initially targeted for RNAi-mediated genome defense. We show that intron-containing mRNA precursors template siRNA synthesis. We identify a Spliceosome-Coupled And Nuclear RNAi (SCANR) complex required for siRNA synthesis and demonstrate that it physically associates with the spliceosome. We find that RNAi target transcripts are distinguished by suboptimal introns and abnormally high occupancy on spliceosomes. Functional investigations demonstrate that the stalling of mRNA precursors on spliceosomes is required for siRNA accumulation. Lariat debranching enzyme is also necessary for siRNA production, suggesting a requirement for processing of stalled splicing intermediates. We propose that recognition of mRNA precursors by the SCANR complex is in kinetic competition with splicing, thereby promoting siRNA production from transposon transcripts stalled on spliceosomes. Disparity in the strength of expression signals encoded by transposons versus host genes offers an avenue for the evolution of genome defense.

Splicing of mouse p53 pre-mRNA does not always follow the “first come, first served” principle and may be influenced by cisplatin treatment and serum starvation

Transcription of a pre-mRNA in eukaryotic cells elongates from the 5' to the 3' end, but intron removal during a pre-mRNA splicing does not always proceed in this orientation. In this study, we identified eight mouse p53 transcripts that retained one or more of introns 6, 7 and 8. The 5' part of intron 9 was also retained while the 3' part was not studied. These intron-containing transcripts, abbreviated as p53-ICTs, were detected at low abundance in many mouse embryonic fibroblasts (MEF) as well as cancer cell lines and tissues, and the highest ratio of these p53-ICTs to the mature p53 mRNA was seen in the normal pancreas. Serum starvation decreased those p53-ICTs that retained introns 6 and 7 but increased the levels of those lacking these introns while the level of the mature p53 mRNA was unaffected. Treatment of several cancer cell lines with cisplatin increased the mature p53 mRNA level but decreased these p53-ICTs. Transfection of p53(-/-) MEF with the p53 cDNA or several p53-ICT mini-genes slightly increased the cell viability and rendered the cells resistant to cisplatin. These data also suggest that p53 pre-mRNA splicing may have multiple orders of intron removal, some of which may not follow the "first come, first served" principle. It remains possible that these p53-ICTs are splicing intermediates existing as a mechanism for the cell to respond more promptly to a demand for more p53 and that p53 protein may be required for a normal life of MEF.

Antisense-targeted immuno-EM localization of the pre-mRNA path in the spliceosomal C complex

A first step in understanding the architecture of the spliceosome is elucidating the positions of individual spliceosomal components and functional centers. Catalysis of the first step of pre-mRNA splicing leads to the formation of the spliceosomal C complex, which contains the pre-mRNA intermediates--the cleaved 5' exon and the intron-3' exon lariat. To topographically locate the catalytic center of the human C complex, we first determined, by DNA oligonucleotide-directed RNAse H digestions, accessible pre-mRNA regions closest to nucleotides of the cleaved 5' splice site (i.e., the 3' end of exon 1 and the 5' end of the intron) and the intron lariat branch point, which are expected to be at/near the catalytic center in complex C. For electron microscopy (EM) localization studies, C complexes were allowed to form, and biotinylated 2'-OMe RNA oligonucleotides were annealed to these accessible regions. To allow localization by EM of the bound oligonucleotide, first antibiotin antibodies and then protein A-coated colloidal gold were additionally bound. EM analyses allowed us to map the position of exon and intron nucleotides near the cleaved 5' splice site, as well as close to the anchoring site just upstream of the branch adenosine. The identified positions in the C complex EM map give first hints as to the path of the pre-mRNA splicing intermediates in an active spliceosomal C complex and further define a possible location for its catalytic center.

Single Molecule FRET Characterization of Pre-mRNA Splicing: Substrate Dynamics during Recognition and Catalysis

The spliceosome is a complex ribonucleoprotein (RNP) that catalyzes the removal of intervening sequences (introns) from coding regions (exons) in eukaryotic pre-mRNAs. The ordered assembly of the spliceosome makes it a compositionally dynamic system where protein and RNA components are shuttled in and out in a highly regulated manner. Furthermore, catalytic activation of the spliceosome requires a dynamic set of ATP dependent RNA-RNA and RNA-protein interactions. Extensive genetic and in vitro biochemical work has been done to define the cis- and trans- acting factors that are required for both steps splicing. On the other hand, the pre-mRNA substrate which acts as both the scaffold for assembly and is a key component of the chemical steps after activation by a yet to be identified spliceosomal catalyst has been wholly ignored thus far. The difficulty in interrogating this system without synchrony and inactivation, thus precluding real time measurements, has proven difficult to overcome. To dissect the kinetic and conformational requirements for pre-mRNA positioning during spliceosome assembly we developed an in vitro single molecule FRET (smFRET) splicing assay in which the position of conserved intronic sequences have been tracked in real-time throughout splicing. The ability to stall the splicing process with mutations in either the pre-mRNA or splicing components we can dissect the role of spliceosome components in regulating pre-mRNA conformations. Additionally, we have utilized an affinity purification technique to determine the relative positions of conserved sequences in isolated splicing intermediates. We have observed reversible conformations changes whose kinetics are affected by ATP and the identity of splice sites.The kinetics of these conformations has implications for proofreading and catalysis that are essential for the maintenance of proper gene expression.

Biogenic mechanisms and utilization of small RNAs derived from human protein-coding genes

Efforts to catalog eukaryotic transcripts have uncovered many small RNAs (sRNAs) derived from gene termini and splice sites. Their biogenesis pathways are largely unknown, but a mechanism based on backtracking of RNA polymerase II (RNAPII) has been suggested. By sequencing transcripts 12-100 nucleotides in length from cells depleted of major RNA degradation enzymes and RNAs associated with Argonaute (AGO1/2) effector proteins, we provide mechanistic models for sRNA production. We suggest that neither splice site-associated (SSa) nor transcription start site-associated (TSSa) RNAs arise from RNAPII backtracking. Instead, SSa RNAs are largely degradation products of splicing intermediates, whereas TSSa RNAs probably derive from nascent RNAs protected by stalled RNAPII against nucleolysis. We also reveal new AGO1/2-associated RNAs derived from 3' ends of introns and from mRNA 3' UTRs that appear to draw from noncanonical microRNA biogenesis pathways.

Branchiostoma floridae has separate healing and sealing enzymes for 5′-phosphate RNA ligation

Animal cells have two tRNA splicing pathways: (i) a 5'-P ligation mechanism, where the 5'-phosphate of the 3' tRNA half becomes the junction phosphate of the new phosphodiester linkage, and (ii) a 3'-P ligation process, in which the 3'-phosphate of the 5' tRNA half turns into the junction phosphate. Although both activities are known to exist in animals, in almost three decades of investigation, neither of the two RNA ligases has been identified. Here we describe a gene from the chordate Branchiostoma floridae that encodes an RNA ligase (Bf RNL) with a strict requirement for RNA substrates with a 2'-phosphate terminus for the ligation of RNAs with 5'-phosphate and 3'-hydroxyl ends. Unlike the yeast and plant tRNA ligases involved in tRNA splicing, Bf RNL lacks healing activities and requires the action of a polynucleotide kinase (PNK) and a cyclic phosphodiesterase (CDPase) in trans. The activities of these two enzymes were identified in a single B. floridae protein (Bf PNK/CPDase). The combined activities of Bf RNL and Bf PNK/CPDase are sufficient for the joining of tRNA splicing intermediates in vitro, and for the functional complementation of a tRNA ligase-deficient Saccharomyces cerevisiae strain in vivo. Hence, these two proteins constitute the 5'-P RNA ligation pathway in an animal organism.

Contribution of DEAH-box protein DHX16 in human pre-mRNA splicing

Studies of mammalian splicing factors are often focused on small nuclear ribonucleoproteins or regulatory RNA-binding proteins, such as hnRNP (heterogeneous nuclear ribonucleoprotein) and SR proteins (serine/arginine-rich proteins); however, much less is known about the contribution of DExD/H-box proteins or RNA helicases in mammalian pre-mRNA splicing. The human DEAH-box protein DHX16 [also known as DBP2 (DEAD-box protein 2)], is homologous with Caenorhabditis elegans Mog-4, Schizosaccharomyces pombe Prp8 and Saccharomyces cerevisiae Prp2. In the present study, we show that DHX16 is required for pre-mRNA splicing after the formation of a pre-catalytic spliceosome. We found that anti-DHX16 antiserum inhibited the splicing reaction in vitro and the antibody immunoprecipitated pre-mRNA, splicing intermediates and spliceosomal small nuclear RNAs. Cells that expressed DHX16 that had a mutation in the helicase domain accumulated unspliced intron-containing minigene transcripts. Nuclear extracts isolated from the dominant-negative DHX16-G724N-expressing cells formed splicing complex B, but were impaired in splicing. Adding extracts containing DHX16-G724N or DHX16-S552L mutant proteins to HeLa cell nuclear extracts resulted in reduced splicing, indicating that the mutant protein directly inhibited splicing in vitro. Therefore our results show that DHX16 is needed for human pre-mRNA splicing at a step analogous to that mediated by the S. cerevisiae spliceosomal ATPase Prp2.

Localization of the Fission Yeast U5.U2/U6 Spliceosome Subunits

The spliceosome is a dynamic macromolecular machine that catalyzes the excision of introns from pre-mRNA to generate protein-coding transcripts. The megadalton-sized spliceosome is composed of four small nuclear RNPs (U1, U2, U5, and U4/U6) and numerous pre-mRNA splicing factors. The formation of an active spliceosome is hypothesized to occur in a stepwise manner requiring the assembly and disassembly of large multiprotein/RNA complexes. A promising structural approach to obtain information about spliceosome complexes is single-particle cryo-electron microscopy (cryo-EM), a powerful technique that is ideal for determining the structures of large dynamic complexes at protein concentrations too low for crystallization. Formerly, our group determined structure of the fission yeast U5.U2/U6 spliceosome complex by cryo-EM. This U5.U2/U6 spliceosome complex contains the U2, U5, and U6 snRNAs, pre-mRNA splicing intermediates, U2 and U5 snRNP proteins, the Nineteen Complex (NTC), and second-step splicing factors. However, the location of these subunits in the complex was not determined. Using antibody labeling and single particle EM we are now localizing these individual subunits within the density map of the U5.U2/U6 spliceosome complex. This work now enables us to propose a structural model for U5.U2/U6 organization.

An Alu-derived intronic splicing enhancer facilitates intronic processing and modulates aberrant splicing in ATM

We have previously reported a natural GTAA deletion within an intronic splicing processing element (ISPE) of the ataxia telangiectasia mutated (ATM) gene that disrupts a non-canonical U1 snRNP interaction and activates the excision of the upstream portion of the intron. The resulting pre-mRNA splicing intermediate is then processed to a cryptic exon, whose aberrant inclusion in the final mRNA is responsible for ataxia telangiectasia. We show here that the last 40 bases of a downstream intronic antisense Alu repeat are required for the activation of the cryptic exon by the ISPE deletion. Evaluation of the pre-mRNA splicing intermediate by a hybrid minigene assay indicates that the identified intronic splicing enhancer represents a novel class of enhancers that facilitates processing of splicing intermediates possibly by recruiting U1 snRNP to defective donor sites. In the absence of this element, the splicing intermediate accumulates and is not further processed to generate the cryptic exon. Our results indicate that Alu-derived sequences can provide intronic splicing regulatory elements that facilitate pre-mRNA processing and potentially affect the severity of disease-causing splicing mutations.

Cwc25 is a novel splicing factor required after Prp2 and Yju2 to facilitate the first catalytic reaction.

Cwc25 has previously been identified to associate with pre-mRNA splicing factor Cef1/Ntc85, a component of the Prp19-associated complex (nineteen complex, or NTC) involved in spliceosome activation. We show here that Cwc25 is neither tightly associated with NTC nor required for spliceosome activation but is required for the first catalytic reaction. The affinity-purified spliceosome formed in Cwc25-depleted extracts contained only pre-mRNA and could be chased into splicing intermediates upon the addition of recombinant Cwc25 in an ATP-independent manner, suggesting that Cwc25 functions in the final step of the first catalytic reaction after the action of Prp2. Yju2 and a heat-resistant factor of unknown identity, HP, have previously been shown to be required for the same step of the splicing pathway. Cwc25, although resistant to heat treatment, is not sufficient to replace the function of HP, indicating that another heat-resistant factor, which we named HP-X, is involved. The requirement of Cwc25 and HP-X for the first catalytic reaction could be partially compensated for when the affinity-purified spliceosome was incubated in the presence of low concentrations of Mn(2+). These results have implications for the possible roles of Cwc25 and HP-X in facilitating juxtaposition of the 5' splice site and the branch point during the first catalytic reaction.

DNA Transcription and tRNA Ligase: the Work of John Abelson

Saccharomyces cerevisiae tRNA Ligase. Purification of the Protein and Isolation of the Structural Gene(Phizicky, E. M., Schwartz, R. C., and Abelson, J. (1986) J. Biol. Chem. 261, 2978–2986)Yeast RNA Ligase Mutants Are Nonviable and Accumulate tRNA Splicing Intermediates(Phizicky, E. M., Consaul, S. A., Nehrke, K. W., and Abelson, J. (1992) J. Biol. Chem. 267, 4577–4582) Saccharomyces cerevisiae tRNA Ligase. Purification of the Protein and Isolation of the Structural Gene (Phizicky, E. M., Schwartz, R. C., and Abelson, J. (1986) J. Biol. Chem. 261, 2978–2986) Yeast RNA Ligase Mutants Are Nonviable and Accumulate tRNA Splicing Intermediates (Phizicky, E. M., Consaul, S. A., Nehrke, K. W., and Abelson, J. (1992) J. Biol. Chem. 267, 4577–4582) John Norman Abelson was born in Grand Coulee Dam, Washington, in 1938. He attended Washington State University and graduated in 1960 with a bachelor's degree in physics. He then moved east to pursue graduate studies at Johns Hopkins University, where he earned a doctorate in biophysics in 1965. After that, Abelson moved again, this time to Cambridge, England, to do a postdoctoral fellowship in the Structural Studies Division at the MRC Laboratory of Molecular Biology. There he worked with Sydney Brenner and Francis Crick studying DNA transcription. In 1968, Abelson returned to the United States to accept a position as assistant professor in the chemistry department at the University of California, San Diego. He was promoted to associate professor in 1973 and professor in 1977. In 1982, Abelson joined the faculty of the California Institute of Technology as a professor of biology. He chaired the Division of Biology and became the George Beadle Professor of Biology in 1991. At Caltech Abelson continued to study transcription, focusing on tRNA precursor splicing and intron excision. In the early 1980s he was able to partially purify tRNA ligase from Saccharomyces cerevisiae (1Greer C.L. Peebles C.L. Gegenheimer P. Abelson J. Mechanism of action of a yeast RNA ligase in tRNA splicing..Cell. 1983; 32: 537-546Abstract Full Text PDF PubMed Scopus (178) Google Scholar). A few years later, he and his colleagues purified the protein to near homogeneity, which is the subject of the first Journal of Biological Chemistry (JBC) Classic reprinted here. Using affinity elution chromatography, they showed that tRNA ligase is a single polypeptide of 90 kDa that also contains polynucleotide kinase and 2′,3′-cyclic phosphate 3′-phosphodiesterase activities. They also determined the amino-terminal sequence of the purified protein and used this information to isolate the gene encoding tRNA ligase by hybridization of an appropriate set of mixed oligonucleotides to a library of yeast DNA. In the second JBC Classic reprinted here, Abelson and his colleagues continue their ligase studies, demonstrating that not only is tRNA ligase critical for tRNA splicing in the yeast cell, but it is also essential for cellular survival. They created a haploid yeast strain carrying a chromosomal deletion of the ligase gene and found that it was viable only if ligase protein was supplied from a plasmid copy of the gene. When synthesis of the plasmid-borne ligase was repressed, the cells accumulated endonuclease-cut, unligated half-molecules and eventually died. Two temperature-sensitive ligase mutant yeast strains also accumulated unligated half-molecules at nonpermissive temperatures. Abelson retired in 2002 and is currently George W. Beadle Professor of Biology, Emeritus at Caltech. He is also president and executive director of Agouron Institute, a non-profit research foundation, which he co-founded in 1978. Abelson also participated in the creation of Agouron Pharmaceuticals, Inc., a small San Diego firm that used rational drug design to develop Viracept, the leading drug used for controlling HIV infections. In recognition of his contributions to science, Abelson has received many awards and honors including the Washington State University (WSU) Distinguished Alumnus Award from the College of Sciences (1993), the WSU Alumni Achievement Award (1995), and the WSU Regents' Distinguished Alumnus Award (2004), as well as election to the National Academy of Sciences (1985), the American Academy of Arts and Sciences (1985), and the American Philosophical Society (2001). He has served as an editor of Methods in Enzymology for the past 15 years and was an assistant editor for Analytical Biochemistry (1980–1987) and a member of the JBC editorial board. He is also a past president of the RNA Society.