THE SPLICEOSOME IS A RIBOZYME

RNA Processing Lecture 1, Biological Regulatory Mechanisms, H.D. Madhani

MAJOR MESSAGES

RNA can fold into stable three-dimensional structures in the absence of protein.

RNA folding has its own grammar that is largely unlike that of protein folding.

Divalent cations play a pivotal role in stabilizing RNA structure.

Phylogenetic comparisons can be used to infer RNA structure.

RNA enzymes, or ribozymes, are widespread

Some ribozymes are metalloenzymes in which Mg++ ions are positioned by ribozyme phosphates to stabilize the transition state of a phosphodiester hydrolysis or phosphodiester trans-esterification reac-tion.

Group II ribozymes, a type of transposable element, and the spliceosome share an evolutionary ances-tor. Introns and the spliceosome evolved after the invasion of a organellar Group II transposon into the nuclear genome of the last eukaryotic common ancestor.

Nuclear pre-mRNA splicing occurs through two sequential phosphodiester trans-esterification reactions that do not result in the net loss or gain of phosphodiester bonds.

The spliceosome, the enzyme responsible for nuclear pre-mRNA splicing, is are assembled from sn-RNPs (small nuclear ribonucleoproteins) and non-snRNP proteins through an pathway characterized massive changes in protein and RNA content prior to formation of the active enzyme.

Dramatic RNA rearrangements characterize spliceosome formation -- to a degree beyond that de-scribed for any other biological system.

The U2-U6 RNA complex coordinates two Mg++ ions to catalyze both chemical steps of pre-RNA splic-ing via a single active site

The most conserved protein in the spliceosome, Prp8, cradles the snRNAs and is evolutionarily related to Group II intron-encoded proteins which function to stabilize the Group II catalytic core.

THE KEY QUESTIONS

1. Evolution: why are eukaryotic genes split into introns and exons?2. Mechanism: how are introns recognized and removed ?

Catalytic RNA: a biological paradigm shiftA little history: Tom Cech observed that nuclei of the cilliate Tetrahymena would synthesize rDNA (tran-scribed by RNA polymerase I) in the presence of alpha-amanatin, a cycic peptide toxin that selectively inhibits RNA polymerases II and III. The rDNA gene contains and intron which he observed was spliced in vitro. Splicing required the addition of guanosine or a guanosine 5’ phosphate, which could be added after transcription (3’ deoxyguanosine was not active). Surprisingly, boiling, protease treatment, phenol extraction or detergent treatment of the transcript before guanosine addition did not not prevent splic-

ing. Indeed, in vitro synthesis of the transcript using a plasmid template and E. coli RNA polymerase produced a splicing-competent RNA.

Cech showed that the guanosine molecule was added to the 5’ end of the intron. Subsequent work identified a guanosine binding site in the IVS. The 3’ OH of the guanosine cofactor attacks the phos-phate at the 5’ splice site releasing the 3’ OH of the 5’ exon and simultaneously attaching the guanosine to the 5’ end of the intron -- a trans-esterification reaction. The guanosine then leaves its binding site and then a guanosine in the intron that immediately precedes the 3’ splice site binds to the guanosine binding site. The 3’ OH of the 5’ exon then attacks the phosphate at the 3’ splice site. The products are the ligated exons and the released IVS.

Splicing requires cations. Mg++ plays a key role in catalysis as shown by metal rescue experiments which which phosphate-bonded oxygen atoms are replaced with sulfur atoms (to produce a phospho-rothioate or phosphorothiolate for non-bridging and bridging oxygens, respectively), reducing the af-finity for Mg++, but increasing the affinity for thiophillic metals such as Mn++ and Cd++. Rescue of a catalysis defect of the sulfur substitued ‘mutant’ by the addition of Mn++ or Cd++ is taken as evidence that the substitued oxygen binds Mg++ in a manner important for activity.

The Tetrahymena IVS was the first RNA enzyme (“ribozyme”) to be identified. Because RNA can be a template for replication and an enzyme, an era of RNA life (the RNA world) may have preceded protein- and DNA-based life.

Like protein enzymes, the IVS folds into a specific three-dimensional structure. The x-ray structure confirms prior models of the guanosine binding site and supported a two-metal mechanism for cataly-sis, mirroring two-metal catalytic centers seen in protein enzymes that catalyze phosphoester hydroly-sis (e.g. EcoRI) and phosphotransfer (RNA and DNA polymerases) reactions.

Several principles guide RNA folding: The A-form double helix is the central secondary structural ele-ment. Folding is then determined by steric constraints and long-range (tertiary) interactions. The latter include interactions between non-helical elements (e.g. a loop base-pairing with another loop -- ‘kissing loop’ structures), between a non-helical region and a helix (e.g. tetraloop-tetraloop receptor), helix-helix (coaxial) stacking, and backbone interactions bridged by cations (usually Mg++ -- note that there are

two types of Mg++ ions in large folded RNAs: structural and catalytic).

Group II introns -- transposons that evolved into the spliceosomeThe Tetrahymena IVS is a member of the “Group I” class of introns which exists in bacteria, bacterio-phage and in eukaryotes. Group II introns, many of which are also self-splicing ribozymes, exist in bacteria and in organelles (mitochondria and chloroplasts). As they share an ancestor with the spliceo-some, they will be considered here.

Group II introns have five or six phylogenetically conserved domains. Domain I, the most complex do-main organizes the folding of the intron and engages in numerous tertiary interactions including binding to the 5’ exon via ‘exon binding sites’, EBS1 and EBS2. Domain IV plays no role in activity, but is often interruped by an open reading from that encodes protein (IEP for ‘intron encoded protein’).

IEP-containing introns require the IEP for splicing. The IEP binds and stabilizes the active conformation of the RNA. These entities are examples of ribonu-cleoprotein enzymes. Domain V is the most con-served domain. Group IIC introns splice by first cata-lyzing a hydrolysis reaction at the 5’ splice site and then a trans-esterification reaction at the 3’ splice site to produce ligated exons and a linear intron. In Group IIA and IIB introns, the nucleophile for the first cata-lytic step is the 2’ OH of an adenosine that is bulged out of a helix in Domain VI.

As for Group I introns, metal rescue experiments sup-port a two-metal catalytic mechanism. The crystal structure of a Group IIC intron supports this model and demonstrates that specific phosphates in Domain V ligands two divalent cations at the 5’ splice site. The structure of Domain V is organized by several tertiary interactions, most prominently three base-triples be-tween between the nearly-invariant “AGC” triad, two residues in a ‘single stranded’ region between Do-mains II and III (J2/3), and one of two residues that bulge out of the domain V helix.

Many Group II introns are mobile elements that have the ability to reverse splice into DNA. The IEP pro-tein of mobile group II introns contains an endonucle-ase domain that cleaves the DNA strand opposite the strand cleaved by reverse splicing. IEPs of mobile introns also contain a reverse transcriptase domain enabling synthesis of a cDNA copy using the 3’OH of the endonuclease-cleaved DNA as a primer.

Thus, an RNA enzyme (the intron) and two protein enzyme activities (endonuclease and reverse tran-scriptase) allow Group II introns to duplicate and spread. In many non-mobile Group II introns, the IEP has lost its catalytic activities, but still binds the intron to enable its splicing activity.

Nuclear pre-mRNA splicing: the basicsNuclear introns were disovered in adenovirus by hybridzationof specific RNAs from infected cells to adenovirus DNA form virions. It was quickly realized that there is not a great deal of sequene information in mammalian introns: a GU dinu-cleotide was present at the beginning of introns and an AG at the end. S. cerevisiae introns (about 2% of genes have introns in this yeast) were found to have more information: a GUAU-GU consensus at the 5’ splice site, a UACUAAC consensus near the 3’ end of the intron and a PyAG at immediately precedent the 3’ splice site. These elements are nearly invariant in S. cerevisiae (which turns out to be the exception in fungi).

Early work produced crude extract systems from HeLa cell nuclear and S. cerevisiae capable of splicing synthetic 32P-radiolabelled pre-mRNA produced by in vitro transcription using phage RNA polymerase. Analysis of the structure of the apparent intermediates and products indicated a two-step reaction which first produces a lariat-intermediate and a cleaved 5’ exon followed by formation of the ligated exons and release of the intron as a lariat-intron.

The spliceosome Velocity sedimentation of yeast in vitro splicing reactions indicates that the lariat-intermediate, cleaved

Exon 1 pGUAUGU UACUAACA yAGp Exon 22’OH

Exon 1 Exon 2 pGUAUGU

UACUAACA

Exon 1

pGUAUGU

UACUAACA yAGp Exon 2

3’OH

5’-2’

5’-2’

Step 1

Step 2

p

yAG 3’OH

5’-exon and excised lariat-intron exist in a 40S particle(s), termed the ‘spliceosome.’ We now know that the yeast spliceosome contains 90 polypeptide chains and 5 snRNAs. The human spliceosome contains ~170 proteins and 5 snRNAs. The prescise function of most of the proteins in the spliceosome is not known.

Five evolutionarily-conserved small nuclear RNAs (snRNAs) are part of the spliceosome and are re-quired for splicing in vitro and in vivo. It was hypothesized that they evolved from an errant Group II intron that invaded the nuclear genome near the time of the birth of the eukaryotic lineage. Each sn-RNA is complexed with a set of proteins to form a ribonucleoprotein (snRNP). While U1 and U2 exist

as individual snRNPs, U4/U5/U6 is found as a single triple-snRNP. U4 and U6 can be separated from U5, but are tightly associated with each other even after deproteinization (heat can be used to separate the two).

Spliceosome assembly basicsAnalysis of the time course of splicing complex assembly in vitro by native gel electrophoresis sug-gested that the snRNA composition of the spliceosome is not static, but rather assembled de novo via a pathway: U1-->U1+U2-->U1+U2+U4/U5/U6-->U2/U5/U6-->catalysis.Inactivation of individual snRNAs by oligonucleotide-directed RNAse cleavage indicated that the prior assembly of U1 and U2 snRNAs onto a pre-mRNA is required for the association of the U4/5/6 triple snRNP with the pre-mRNA.

Roles for Watson-Crick base-pairing in intron recognition by U1 and U2 snRNAs U1 displays a complementarity to the 5’ splice site consensus and U1 snRNP can bind and protect the 5’ splice site in vitro from nuclease digestion.

Base-pairing disruption-restoration (‘compensatory base-pair”) experiments demonstrate that this in-teraction is funcitonal in vivo. U2 snRNP can protect sequences around the branchpoint. Genetic suppression Watson-Crick base-pairing between the evolutionarily invariant GUAGUA sequence in U2 snRNA and the UACUAAC sequence in yeast introns is required for splicing in vivo. For organisms with weaker branchpoint consensus sequences, a protein, U2AF (U2 -auxiillary factor) helps U2 snRNP bind to the branchpoint by recognizing a downstream pyrimidine tract.

The 5’ splice site is recognized a second time, this time by U6Early studies in yeast identified a mutation in the 5’ splice site that produced an unexpected phenotype: mutation of the G at position 5 of the 5’ splice site to an A, not only blocked cleavage at the 5’ splice site, it activated cleavage at a nearby sequence that had little resemblance to a 5’ splice site. They reasoned that the 5’ splice site was recognized more than once, first by U1 and then by something else.

Psoralen crosslinking studies and genetic suppression studies demonstrate that a conserved sequence in U6 snRNA just upstream of where U6 base-pairs with U4 base-pairs with 5’ splice site. Furthermore the analysis demonstrated that the U6-5’ splice site pairing and not the U1-5’ splice site pairing actually determines where 5’ cleavage occurs.

The branchpoint is recognized by a protein before it base-pairs with U2

Branchpoint binding protein (BBP/SF1) is a protein that specifcally recognizes the UACUAAC se-quence. It binds cooperatively to RNA with U2AF. A U2 snRNP protein, SF3b155, directly binds U2AF.

“Committment complex 1” (CC1) contains pre-mRNA, U1 snRNP, BBP/SF1 and is assembled on syn-thetic, labelled pre-mRNA in using extracts in which U2 has been inactivated. CC1 assembled onto labelled pre-mRNA can be “chased” to mRNA. This can be accomplished by addition of an excess of unlabelled pre-mRNA and an extract containing active U2 snRNP.

Summary of intron recognition events (so far)

RNA rearrangements and the catalytic activation of the spliceosome

Exon 1 pGUAUGU UACUAACA PPT-yAGp Exon 22’OH

CAUUCAUApppG

GAGACA

BBP/SF1

Exon 1 pGUAUGU UACUACA PPT-yAGp Exon 2

2’OH

AUGAUGU

A

U2AF

U2

U1

U6 5’3’ 5’3’

3’U2AF

The structure of the U4-U6 interaction was revealed by the cloning of the S. cerevisiae U6 snRNA gene and using phylogenetic comparison to identify compensatory base changes.

Genetic analysis showed that a region of U6 snRNA that base-pairs with U4 was much more sensitive to point mutations than the complementary residues in U4. This result and the the apparent loss of U1

and U4 from splicing complexes prior to the appearance of intermediates and products suggested that U6 might engage in new interactions that are mutually-exclusive with the U4-U6 interaction.

Guesswork and genetic analysis revealed that this region of U6 initially base-paired to U4 forms a novel

interaction with U2 adjacent to where U2 base-pairs with the intron branch point.

Combined with an intramolecular interaction found in the free U6 snRNP (the intramolecular stem-loop or ISL) and another U2-U6 interaction (helix II), a picture emerges of a presumptive RNA active site. The structure bears a strong resemblance to Domains V and VI of Group IIA/B introns including the AGC triad.

These results implies at least three RNA helices were disrupted during the catalytic activation of the spliceoseosome: U4-U6 stem I, U5-U6 stem II, and the 5’ stem-loop of U2 snRNA.

U6 is the spliceosomal ribozyme

The ability to reconstitute splicing after degradation of endogenous U6 snRNA by the addition of syn-thetic U6 snRNA combined with phosphothioate/phosphorothiolate metal rescue experiments demosn-trated that the U2-U6 complex functions to position two catalytic Mg++ ions. Moreover, the phosphates that ligand Mg++ occur in positions analogous to those that ligand Mg++ in Domain V of the Group IIC crystal structure.

A high resolution cryoEM structure of a likely post-catalytic spliceosome (the S. pombe intron-lariat spliceosome or “ILS”) confirms that the U2-U6 complex coordinates two divalent cations to produce a Group II-like active site.

Prp8, the most conserved protein in the spliceosome, is related to IEPs

Crosslinking and genetic suppression studies point to Prp8, the most conserved protein in the spliceo-some as being a component of the spliceosome closely associated with the snRNAs and the pre-mRNA substrate. The crystal structure of Prp8, which is part of U5 snRNP indicates that it is highly related to IEPs. Prp8 contains a reverse transcriptase domain, an endonuclease domain and (unlike known IEPs) an RNase H domain. All appear to be catalytically inactive. This finding supports the hypothesis that the spliceosome evolved from a once-mobile Group II intron, presumably near the time of the birth of the eukaryotic lineage.

Reviews

Madhani, H.D., and Guthrie, C. (1994a). Dynamic RNA-RNA interactions in the spliceosome. Annual review of genetics 28, 1-26.

Nguyen, T.H., Galej, W.P., Fica, S.M., Lin, P.C., Newman, A.J., and Nagai, K. (2016b). CryoEM struc-tures of two spliceosomal complexes: starter and dessert at the spliceosome feast. Current opinion in structural biology 36, 48-57.

Peters, J.K., and Toor, N. (2015). Group II intron lariat: Structural insights into the spliceosome. RNA biology 12, 913-917.

Staley, J.P., and Guthrie, C. (1998). Mechanical devices of the spliceosome: motors, clocks, springs, and things. Cell 92, 315-326.

Will, C.L., and Luhrmann, R. (2011). Spliceosome structure and function. Cold Spring Harbor perspec-tives in biology 3.

Zimmerly, S., and Semper, C. (2015). Evolution of group II introns. Mobile DNA 6, 7.

Primary references

Adams, P.L., Stahley, M.R., Kosek, A.B., Wang, J., and Strobel, S.A. (2004). Crystal structure of a self-splicing group I intron with both exons. Nature 430, 45-50.

Berglund, J.A., Chua, K., Abovich, N., Reed, R., and Rosbash, M. (1997). The splicing factor BBP inter-acts specifically with the pre-mRNA branchpoint sequence UACUAAC. Cell 89, 781-787.

Brody, E., and Abelson, J. (1985). The “spliceosome”: yeast pre-messenger RNA associates with a 40S complex in a splicing-dependent reaction. Science 228, 963-967.

Brow, D.A., and Guthrie, C. (1988). Spliceosomal RNA U6 is remarkably conserved from yeast to mam-mals. Nature 334, 213-218.

Butcher, S.E., and Pyle, A.M. (2011). The molecular interactions that stabilize RNA tertiary structure: RNA motifs, patterns, and networks. Accounts of chemical research 44, 1302-1311.

Cech, T.R., Zaug, A.J., and Grabowski, P.J. (1981). In vitro splicing of the ribosomal RNA precursor of Tetrahymena: involvement of a guanosine nucleotide in the excision of the intervening sequence. Cell 27, 487-496.

Chan, R.T., Robart, A.R., Rajashankar, K.R., Pyle, A.M., and Toor, N. (2012). Crystal structure of a group II intron in the pre-catalytic state. Nature structural & molecular biology 19, 555-557.

Couto, J.R., Tamm, J., Parker, R., and Guthrie, C. (1987). A trans-acting suppressor restores splicing of a yeast intron with a branch point mutation. Genes & development 1, 445-455.

Domdey, H., Apostol, B., Lin, R.J., Newman, A., Brody, E., and Abelson, J. (1984). Lariat structures are

in vivo intermediates in yeast pre-mRNA splicing. Cell 39, 611-621.

Fica, S.M., Mefford, M.A., Piccirilli, J.A., and Staley, J.P. (2014). Evidence for a group II intron-like cata-lytic triplex in the spliceosome. Nature structural & molecular biology 21, 464-471.

Fica, S.M., Tuttle, N., Novak, T., Li, N.S., Lu, J., Koodathingal, P., Dai, Q., Staley, J.P., and Piccirilli, J.A. (2013). RNA catalyses nuclear pre-mRNA splicing. Nature 503, 229-234.

Galej, W.P., Oubridge, C., Newman, A.J., and Nagai, K. (2013). Crystal structure of Prp8 reveals active site cavity of the spliceosome. Nature 493, 638-643.

Hang, J., Wan, R., Yan, C., and Shi, Y. (2015). Structural basis of pre-mRNA splicing. Science 349, 1191-1198.

Keating, K.S., Toor, N., Perlman, P.S., and Pyle, A.M. (2010). A structural analysis of the group II intron active site and implications for the spliceosome. Rna 16, 1-9.

Kruger, K., Grabowski, P.J., Zaug, A.J., Sands, J., Gottschling, D.E., and Cech, T.R. (1982). Self-splic-ing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell 31, 147-157.

Lesser, C.F., and Guthrie, C. (1993). Mutations in U6 snRNA that alter splice site specificity: implica-tions for the active site. Science 262, 1982-1988.

Lin, R.J., Newman, A.J., Cheng, S.C., and Abelson, J. (1985). Yeast mRNA splicing in vitro. The Journal of biological chemistry 260, 14780-14792.

Madhani, H.D., and Guthrie, C. (1992). A novel base-pairing interaction between U2 and U6 snRNAs suggests a mechanism for the catalytic activation of the spliceosome. Cell 71, 803-817.

Madhani, H.D., and Guthrie, C. (1994b). Randomization-selection analysis of snRNAs in vivo: evidence for a tertiary interaction in the spliceosome. Genes & development 8, 1071-1086.

Mount, S.M., Pettersson, I., Hinterberger, M., Karmas, A., and Steitz, J.A. (1983). The U1 small nuclear RNA-protein complex selectively binds a 5’ splice site in vitro. Cell 33, 509-518.

Nguyen, T.H., Galej, W.P., Bai, X.C., Oubridge, C., Newman, A.J., Scheres, S.H., and Nagai, K. (2016a). Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 A resolution. Nature.

Nguyen, T.H., Galej, W.P., Bai, X.C., Savva, C.G., Newman, A.J., Scheres, S.H., and Nagai, K. (2015). The architecture of the spliceosomal U4/U6.U5 tri-snRNP. Nature 523, 47-52.

Parker, R., and Guthrie, C. (1985). A point mutation in the conserved hexanucleotide at a yeast 5’ splice junction uncouples recognition, cleavage, and ligation. Cell 41, 107-118.

Parker, R., Siliciano, P.G., and Guthrie, C. (1987). Recognition of the TACTAAC box during mRNA splic-ing in yeast involves base pairing to the U2-like snRNA. Cell 49, 229-239.

Piccirilli, J.A., Vyle, J.S., Caruthers, M.H., and Cech, T.R. (1993). Metal ion catalysis in the Tetrahymena ribozyme reaction. Nature 361, 85-88.

Robart, A.R., Chan, R.T., Peters, J.K., Rajashankar, K.R., and Toor, N. (2014). Crystal structure of a eukaryotic group II intron lariat. Nature 514, 193-197.

Ruskin, B., Krainer, A.R., Maniatis, T., and Green, M.R. (1984). Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro. Cell 38, 317-331.

Ruskin, B., Zamore, P.D., and Green, M.R. (1988). A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly. Cell 52, 207-219.

Toor, N., Keating, K.S., Taylor, S.D., and Pyle, A.M. (2008). Crystal structure of a self-spliced group II intron. Science 320, 77-82.

Wan, R., Yan, C., Bai, R., Wang, L., Huang, M., Wong, C.C., and Shi, Y. (2016). The 3.8 A structure of the U4/U6.U5 tri-snRNP: Insights into spliceosome assembly and catalysis. Science.

Wassarman, D.A., and Steitz, J.A. (1992). Interactions of small nuclear RNA’s with precursor messen-ger RNA during in vitro splicing. Science 257, 1918-1925.

Yan, C., Hang, J., Wan, R., Huang, M., Wong, C.C., and Shi, Y. (2015). Structure of a yeast spliceo-some at 3.6-angstrom resolution. Science 349, 1182-1191.

Zamore, P.D., Patton, J.G., and Green, M.R. (1992). Cloning and domain structure of the mammalian splicing factor U2AF. Nature 355, 609-614.