Spliceosome

Miro Brajenovic

Introduction:
The occurrence of introns in nuclear precursor RNAs (pre-mRNAs) is widespread in eukaryotes, and the splicing process that removes them is basically the same in yeast as it is in higher eukaryotes. Splicing takes place in a very large, multi-component complex, the spliceosome. Over the past 10 years, significant progress has been made in the understanding of how RNA-binding factors may facilitate splice-site selection and spliceosome assembly, and confer fidelity to the pre-mRNA splicing reaction. The formation of a spliceosome complex takes place in the RNA editing proces, where a newly synthesized RNA molecule, the primary transcript, is established. A primary transcript for a eukaryotic mRNA typically contains sequences encompassing one gene, and it is highly heterogeneous (hnRNA) in length (2000-20000 nucleotides). This is much larger than expected from the known size of protein. However, the sequences encoding the polypeptide are usually not contiguous.

The coding sequence (exon) is interrupted by noncoding tracts called introns. In a process called splicing, the introns are removed from the primary transcript and the exons joined to form a contiguous sequence specifying a functional peptide. The base sequences of thousands of intron-exon junctions of RNA transcripts are known, and they are revealing. These sequences in eukaryotes from yeast to mammals have a common structural motif: the base sequence of an intron begins with GU and ends with AG. Thus, the consensus sequence at the 5’ splice site of vertebrate introns is AGGUAAGU. Introns have an important internal site located between 20 and 50 nucleotides upstream of the 3’ splice site; it is called the branch site. Also, eukaryotic m-RNAs are modified at each end. A structure called a cap is added at the 5’ end, and a polymer of 20-230 adenylate residues, poly (A), is added to the 3’ end (Staley et al, 1998).

Splicing of free mRNA precursors does not occur. Rather, as soon as RNA polymerase II produces a sufficient length of RNA, the nascent transcript associates with specific proteins to form the heterogeneous nuclear ribonucleoprotein complex (hnRNP). The complex is composed of 8 to 10 proteins, with a molecular weight (in mammals) ranging from 32,000 to 120,000. These proteins bind to approximately 500 nucleotides of RNA. The hnRNPs facilitate the processing of the hnRNAs and their transport to different regions of the nucleus. In the cytoplasm a mature mRNA associates with an entirely different set of proteins. Also, hnRNPs may protect the primary transcript from endogenous nucleases and inhibit the formation of secondary structures. In fact, according to Staley et al., one of the proteins of the complex is a helix-destabilizing protein (HD protein). HD protein denatures double stranded RNA structures, such as hairpins, and HD inhibits the formation of secondary structures (Staley et al, 1998).

Splicing requires the action of specialized RNA-protein complexes containing a class of eukaryotic RNAs called small nuclear RNAs (snRNAs). Five snRNAs, U1, U2, U4, U5, U6, are involved in the splicing reactions. They are found in abundance in the nuclei of many eukaryotes, and they range in size from 106 (U6) to 185 (U2) nucleotides (Table 1.). These snRNAs are complexed with specific proteins, (more than 50), to form particles called small ribonucleoproteins (snRNPs). According to Steitz, the 5’ end of one of these snRNAs , U1-snRNAs (so called because it is a member of a U-rich subfamily of snRNAs), is partially complementary to the consensus sequence of 5’ splice junctions. Addition of the U2, U4, U5, and U6 snRNP leads to formation of a complex called the spliceosome where the actual splicing reaction occurs (Misteli et al, 1997).

Table 1. Small ribonuclearprotein particles (snRNPs) involved in the splicing of mRNA precursors .

These aforementioned mRNA splicing factors within the mammalian cell nucleus are concentrated in 20-40 distinct domains called speckles. According to Misteli et al. dynamic properties of splicing factors were observed in nuclei of living cells. They showed that speckles are highly dynamic structures that respond specifically to activation of nearby genes. When single genes are transcriptionally activated in living cells, splicing factors leave speckles on peripheral extensions and accumulate at the new sites of transcription. Misteli et al. tested how the dynamic events are related to RNA polymerase II (RNA II) activity. Peripheral movements were strictly dependent on ongoing RNA II transcription. To prove the RNA II involvement in that process, Misteli et al. used RNA II inhibitor a-amanitin to see if any changes would be detected. But neither peripheral extensions nor disassociating particles were observed. Only 2% of speckles showed peripheral movements, in comparison with 82% when the inhibitor was not present. They have shown that pre-mRNA splicing factors are dynamic within the interphase and are rapidly recruited from speckles to sites of transcription after gene activation. The observations directly demonstrated that one function of speckles is to supply pre-mRNA splicing factors to sites of active transcription (Misteli et al, 1997).

According to Chiara et al., both spliseosome and ribosome are energy-consuming machines, but neither requires ATP hydrolysis for the basic chemical reaction promoted by each machine. No NTP hydrolysis is required for the chemistry of splicing, because catalysis proceeds by two phosphoryl transfer reactions (two transesterification reactions explained later in text). According to Staley et al., the spliceosome is a very dynamic machine that is building anew on each pre-mRNA substrate. Each round of splicing requires the ordered binding and release of snRNPs. Although the earliest assembly step is energy independent (Chiara et al, 1996), all subsequent assembly steps require NTP hydrolysis. Coincident with many of these steps, RNA helices are formed and disrupted. Intriguingly, each ATP-dependent step requires one or more proteins of a superfamily of ATPases that share sequence similarity with canonical DNA helicases, proteins that function as processive motors. This similarity has suggested that the spliceosomal members may consume ATP to unwind RNA. Also, Staley et al. concluded that the precise timing of the RNA rearrangements suggests that these RNA-dependent ATPases proofread steps of splicing (Staley et al, 1998).

Hetzer et al. explained how the rearrangements in the spliceosome assemble a catalytically active spliceosome. The U2 snRNP and then the U4/U6 U5 triple snRNP bind to the arranged spliceosome complex. Then U1 and U4 are destabilized, and the spliceosome is activated for catalysis. This assembly order is the same in all organisms from yeast to mammals. At the molecular level, assembly begins with U1 snRNP recognizing the 5’ splice site on pre-mRNA. This U1 RNA contains a sequence that is complementary to this splice site, and the binding of U1 snRNP to an mRNA precursor protects a 15-nucleotide region at the 5’splice site from digestion. U1 is not required during the transesterification reactions. This interaction is switched subsequently for a mutually exclusive base-pairing interaction with U6. It means that U6 now binds to that reagion instead of U1. The branch point sequence is recognized early by BBP, the branch point binding protein, in a sequence-specific fashion. During the assembly of the spliceosome, U2 snRNP binds to the branch site in the intron instead of BBP.

Thus, subsequent recognition of the branch point region by base pairing with U2 snRNA is also mutually exclusive, U2 binds to the branch point instead of BBP. The association of U1 and U2 brings together the 5’ and 3’ ends of the intron, and U1 is enabled to pair with the 3’ splice site. This complex of U1, U2, and the mRNA precursor is joined by a preassembled U4-U5-U6 complex to form a complete splicesome (Hetzer et al, 1997). The triple U4-U5-U6 is believed to escort U6 to the spliceosome to base pair with U4 via stems I and II. In yeast, this interaction is remarkably stable, but despite this stability, both stems of the U4/U6 interaction are disrupted as the spliceosome undergoes extensive remodeling and becomes activated for catalysis. U4 is not required for catalysis and it does not fall off, it is deactivated. The stem II region of U6, once freed from U4, folds on itself to form an intramolecular U6 3’ stem/loop. The stem I region of U6, once freed, base pairs with U2 snRNA, forming U2/U6 helix I. Finally, the U2 5’ stem/loop is responsible for formation of U2/U6 helix II (Staley et al, 1998).

According to Chiara et al. U2 and U6 snRNAs are probably involved in forming the catalytic center of the spliceosome. Having executed these RNA rearrangements, the catalytically active spliceosome is competent to carry out the first chemical step. The first transesterificiation reaction begins with the cleavage of phosphodiester bond between the upstream exon (exon1) and the 5’ end of the intron. The attacking group in this reaction is the 2’-OH of an adenylate residue in the branch site. A 2’,5’-phosphodiester bond is formed between this A residue and the 5’terminal phosphate of the intron. This adenylate residue is also joined to two other nucleotides by normal 3’,5’ phosphodiester bonds. Chiara et al. noticed that a branch is generated at this site and a lariat intermediate is formed.

In the second reaction, the 3’-OH terminus of exon1 then attacks the phosphodiester bond between the intron and exon2. Exons 1 and 2 become joined , and the intron is released in lariat form together with the U2, U5, U6 bound to it. In the first reaction, RNA directs the alignment of the splice site and catalyses the reaction. The second reaction is the unwinding of the RNA duplex intermediates and inducing the release of ribonucleoproteins from mRNA precursors and products. Until the two exons are joined, the products of the first reaction are held together by the spliceosome (Chiara et al, 1996).

Laggerbauer et al. detected physical contacts between the splicesome and the RNA substrate in regions between the branch point/polypyrimidine tract and the 3’ splice site that are not important for splicing. To block step 2 of the splicing process hairpin structures were inserted between the branch point and the 3’ splice site and methylene blue was used to crosslink the protein with dsRNA. By using this approach Laggerbauer et al. detected a 116-kDa crosslinked protein involved in step 2 of splicing. The protein was identified as the 116-kDa U5 snRNP protein. The crosslinking characteristics of U5 are consistent with its role in locating the 3’ splice site AG prior to step 2 of splicing (Laggerbauer et al, 1997).

According to O’Keefe and Newman, the U5 snRNA loop 1 in yeast interacts with the 5’ exon before the first step of pre-mRNA splicing and with 5’ and 3’ exons following the first step. These U5-exon interactions are proposed to hold the exons in the correct orientation for the second step of splicing. The experiment in vitro indicated that U5 loop 1-5’ exon interactions are not necessary for the first catalytic step but are critical for the second step in yeast splicesomes. O’Keefe and Newman systematically made deletion and insertion (mutations) in loop 1 and then monitored splicing activity and loop-exon interactions by cross-linking. Their results showed that a single nucleotide deletion or insertion (mutation) in loop 1 permitted both steps of splicing. Also, larger mutations allowed the first step, but progressively inhibited the second step. Thus, if more base pairs were deleted, there was more inhibition of the second step. The analysis of the loop 1 insertions and deletions by cross-linking revealed that inhibition of the second catalytic step resulted from misalignment of the 5’ and 3’ exons. Their data indicated that the size of loop 1 is critical for proper alignment of the exons for the second catalytic step of splicing and that the 3’ exon is positioned on the loop independently of the 5’ exon (O’Keefe and Newman, 1998).

According to Hetzer et al. restoration of the second-step of catalysis is governed by a terminal loop structure and by the potential of the loop sequences to anchor the exon sequences. In those sequences central U residues of U5 snRNA are involved in this reaction. First, U5 interacts with exon sequences in the 5’ splice site. The next step is the ATP-driven melting of the base pairing between U1 and the 5’ splice site. U1 leaves the spliceosome, and the U5 extends its pairing with the mRNA precursor to include the 5’ splice site. Also they found that the disruption of the pairing between U4 an U6 sets the stage for splicing by unleashing the catalytic activity of U6 (Hetzer et al, 1997).

Staley et al. have discussed the postcatalytical rearrangements and recycling. After the spliceosome ligates the exons and produces mRNA, it must liberate the mRNA to be exported. Furthermore, Staley et al. compared spliceosome with a machine, which, after performing repetitive tasks must be reconfigured to allow a new round of splicing. The snRNP-bound lariat intron must be disassembled, allowing the lariat form to be degraded and the snRNPs to be recycled. Release of mRNA, disassembly, and recycling all involve extensive RNA:RNA rearrangements. The base pairing of U6 to the 5’ splice site, U2 to branch point, and U5 to the exons must be severed. Moreover, the mutually exclusive pairing involving U2, U6, and U4 must be disrupted to return to their original conformation (Staley et al, 1998).

The discovery of interrupted genes and mRNA splicing was greeted with amazement 20 years ago. 10 years ago it was discovered that that the splicing apparatus included five snRNPs and more than 50 other proteins. So, with regard to the short time period between these two discoveries, it seems that soon we would be able to know more about the assembling of spliceosome at the molecular level. The mRNA splicing factors are found within the mammalian cells in domains called speckles, that supply pre-mRNA splicing factors to sites of active transcription. The spliceosome is a catalytically active complex, which does not need energy for the earliest assembling, but all subsequent assembly steps require NTP hydrolysis. Spliceosome assembly, rearrangements, and disassembly requires ATP. The spliceosome is a very complex molecular assembly, and the splicing process includes two main steps and many additional substeps. After splicing, the complex must be disassembled to allow the lariat intron to be degraded and the snRNP to be recycled. Since each of the rearrangements involves both RNA unwinding and annealing, it is likely that many more annealing factors remain to be identified. I would suggest that one of the future goals could be the identification of whether the targets of each unwinding and "rewinding" proteins are RNA, protein, or RNP.

Chiara, Maria Dolores, and Reed, Robert. (1996). A two-step mechanisms for 5’ and 3’ splice-site pairing. Nature. v375 p510(4).

Hetzer, Martin, Wurzer, Gabriele, Schweyen, J., and Mueller, Manfred. (1997). Trans-activation of group II intron splicing by nuclear U5 snRNA. Nature. v385, p357(5).

Laggerbauer, B., Liu, Z.R., Luhram, R., and Smith, C.W. (1997). Crosslinking of the U5 snRNP-specific 116-kDa protein to RNA hairpins that block step 2 of splicing. v3 p1207-1219.

Misteli, Tom, Caceres, Javier and Spector, David. (1997). The dynamics of a pre-mRNA splicing factors in living cells. Nature. v387 p523(5).

O’Keefe, R.T. and Newman, A.J. (1998). Functional analysis of the U5 snRNA loop 1 in the second catalytic step of yeast pre-mRNA splicing. v17 p565-574.

Staley, Jonathan, P., and Guthrie, Christine. (1998) Mechanical devices of the spliceosome: Motors, Clocks, Springs, and Things. Cell. V92 p315-326.

Copyright © 1998 Miro Brajenovic and Koni Stone

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