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PERSPECTIVE

Ironing out the kinks: splicing and translation in bacteria

Sarah A. Woodson1 Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742-2021 USA

In eukaryotic cells, RNA processing is physically sepa- groups (Pyle et al. 1992). Recognition of the 3Ј splice rate from synthesis. As nuclear export of un- involves several weak interactions, including a 2-bp spliced RNA is restricted, only mature messages are nor- stem between the 3Ј end of the intron and in mally exposed to the translational machinery. In bacte- the intron core (Burke et al. 1990). The folded structure ria, however, splicing must be coordinated with the of the RNA depends on coordination of magnesium ions, translation of nascent transcripts. These two processes which are required for self-splicing (Cech and Herschlag make very different demands on the RNA substrate: 1996). Long-range interactions, such as base-pairing be- Splicing of autocatalytic introns requires that the 5Ј and tween hairpin loops, or tetraloop–helix receptor interac- 3Ј splice sites be brought together as part of an elaborate tions, also stabilize the tertiary structure of the catalytic tertiary structure, whereas translation requires that the core (for review, see Brion and Westhof 1997). mRNA be relatively free of secondary structure. None- Recent experiments on the folding kinetics of group I theless, introns have been found in highly expressed introns, as well as experiments carried out in the 1970s in eubacteria, bacteriophages, mitochondria, and on tRNA, have begun to tease out the mechanisms by chloroplasts (for review, see Burke 1988). Clearly, there which reach a biologically active conformation must be some means of balancing splicing of bacterial (for review, see Draper 1996; Brion and Westhof 1997). introns with cotranscriptional translation. In this issue, Small hairpins can form in 10–100 µsec, and tRNAs can Semrad and Schroeder (1998) provide the surprising an- fold within milliseconds (Draper 1996). In contrast, swer that splicing of a group I intron from phage T4 is larger RNAs fold in stages over much longer periods of facilitated by translation of the upstream open reading time. The tertiary structure of the P4–P6 domain of the frame (ORF). This enhancement of splicing is achieved Tetrahymena group I intron, which can fold indepen- by modulating the long-range conformation of the pre- dently, appears in a few seconds (Sclavi et al. 1997). The mRNA. Their results provide useful analogies for the core of the intron, however, takes several minutes to coupling of eukaryotic pre-mRNA splicing with tran- become completely folded (Zarrinkar and Williamson scription. 1994; Banerjee and Turner 1995). Bacterial mRNAs exclusively contain group I or group The very slow folding of longer RNAs arises, in part, II introns, and the three group I introns that are present from their tendency to form many alternative secondary in phage T4 are all able to self-splice in vitro (for review, structures. As RNA secondary structure is stable, incor- see Belfort 1990). The introns are found in genes encod- rect base pairs have the potential to trap the molecule in ing thymidylate synthase (td), ribonucleotide reductase inactive conformations that can persist for relatively (nrdB) (Belfort 1990), and anaerobic ribonucleotide re- long periods of time (for review, see Herschlag 1995; ductase (sunY, or nrdD) (Young et al. 1994). In addition Thirumalai and Woodson 1996; Brion and Westhof to sequences that provide the necessary functions of self- 1997). These metastable states may be quite dissimilar splicing, td and sunY in trans contain internal ORFs that to the final structure. Therefore, the folding process it- encode double-stranded DNA endonucleases (Belfort self can result in RNA populations with different levels 1989). The endonucleases trigger homing, or site-specific of biological activity. Misfolding of RNA has been movement of the intron sequences to intronless alleles. shown to inhibit activity and spliceosome as- Self-splicing requires that the intron RNA fold into a sembly (Goguel et al. 1993; Zavanelli et al. 1994; Uhlen- unique secondary and tertiary structure (Cech and Her- beck 1995). As discussed below, competition between schlag 1996). The central core of this structure is highly metastable RNA conformations can also serve as a nor- conserved among group I introns and contains the active mal mechanism by which activity is regulated. site where the transfer of phosphodiester bonds takes Estimates of in vivo splicing rates are 10- to 50-fold place. A helix containing the 5Ј splice site docks into the faster than in vitro self-splicing (Brehm and Cech 1983; active site via hydrogen bonds with its ribose 2Ј hydroxyl Zhang et al. 1995). This disjunction between in vitro and in vivo activity of catalytic RNAs implies that kinetic 1E-MAIL [email protected]; FAX (301) 314-9121. folding traps are normally overcome in the .

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Woodson that facilitate splicing of group I and group II introns in fungal mitochondria were first identified as the result of splicing-deficient (Gampel et al. 1989; Lam- bowitz and Perlman 1990). Biochemical experiments have established that these proteins promote splicing by stabilizing the correctly folded structure of the intron RNA. Neurospora crassa CYT-18 mitochondrial tRNA synthetase binds to a conserved region of the catalytic core and maintains the alignment of two double helices that form part of the active site (Caprara et al. 1996). The yeast protein CBP2 not only stabilizes the core of the intron, but also promotes docking of the splice site helix with the catalytic core (Weeks and Cech 1995). Another class of RNA-binding proteins accelerates the resolution of misfolded RNA structures under certain conditions in vitro, in a manner analogous to protein chaperones (Herschlag 1995). These proteins, which in- clude hnRNP A1 and HIV nucleocapsid protein, prefer- Figure 1. Translation enhances splicing of the td group 1 in- entially bind single-stranded RNA with low sequence tron from phage T4 in vivo. Recognition of the 3Ј splice site (3Ј specificity and promote nonspecific reannealing of RNA SS) is inhibited by base-pairing of the 5Ј with the 3Ј end of duplexes (Herschlag 1995). Ribosomal protein S12 en- the intron (green); this pairing is prevented by ribosomes bound upstream (Semrad and Schroeder 1998). Ribosomes may also hances an intermolecular splicing reaction of a phage T4 stabilize the folded structure of the intron. Translation of the intron and increases the turnover rate of a hammerhead pre-mRNA terminates at a stop codon (red) after the 5Ј splice ribozyme (Coetzee et al. 1994). It is not yet clear whether site (5Ј SS). The internal double-strand DNA endonuclease I-Tev any of these proteins function as true RNA chaperones I is indicated in blue; the core of the td intron is omitted for in vivo. However, a cold shock protein from Escherichia clarity. Adapted from Belfort (1990). coli has also been shown to have RNA chaperone activ- ity in vitro (Jiang et al. 1997), suggesting that its function is to compensate for overstabilization of RNA secondary mRNA from splicing, they introduced stop codons up- structures at low temperatures. stream of the 5Ј splice site. Surprisingly, they found that The fact that introns are found in the highly stream- splicing in vivo was reduced to nearly undetectable lev- lined of bacteriophages is somewhat surprising, els, even in the absence of antibiotics (Semrad and especially as intronless phage suffer no loss in viability Schroeder 1998). Splicing was restored partially by a sup- (Belfort 1989). In part, their persistence in the T4 pressor tRNA, as judged by both analysis of td RNA and is ensured by the two homing endonucleases. However, the ability of strains to grow on minimal media lacking the presence of related introns in other T-even phages, thymine. These observations suggested that translation and coordination of splicing and expression of the hom- itself was required for efficient splicing in vivo. ing endonucleases with the phage life cycle, suggest a Reduced splicing of transcripts containing upstream long period of coadaptation (Belfort 1990). What factors, stop codons was not a result of a change in mRNA sta- then, facilitate splicing of phage T4 pre-mRNA? bility, as stop codons had no effect on levels of td mRNA T4 td is expressed early in infection from a lacking the intron. Neither was the peptide product re- upstream of the external ORF (Belfort 1990). Because the quired for in vivo splicing, as tandem frameshift muta- intron interrupts the reading frame, splicing must occur tions that alter the peptide sequence while maintaining before translation to produce full-length td protein (Fig. the continuity of the ORF did not inhibit splicing. What 1). However, protein synthesis in E. coli begins shortly did matter, however, was the position of the stop codon. after . As the td intron is 1016 bases long, In general, splicing levels increased when stop codons ribosomes reach the 5Ј splice site long before the 3Ј were placed close to the 5Ј splice site, and decreased splice site is transcribed. Movement of ribosomes into when they were introduced farther upstream. the interior of the intron inhibits splicing by disrupting The observation that splicing activity varied with the the folded structure of the RNA (O¨ hman-Hede´n 1993). In position of the stop codon indicated that a folding defect td, this is prevented by a stop codon just after the 5Ј in the pre-mRNA could be responsible for inhibition. splice site (Belfort 1989). Termination of protein synthe- Moreover, the requirement for translation suggested that sis at this stop codon produces a truncated protein ribosomes altered the RNA folding pattern. To test this (NH2–TS) with no known function. idea, proteins that are known to enhance splicing of the Clearly, expression of td requires coordination among td intron were overexpressed in strains carrying the td transcription, splicing, and translation. To address this gene constructs. Splicing of pre-mRNAs containing up- issue, Semrad and Schroeder began by setting out to in- stream stop codons were improved by overexpression of vestigate another phenomenon, namely, the inhibition both S12, which enhances splicing in a nonspecific man- of group I splicing by aminoglycoside antibiotics (von ner (Coetzee et al. 1994), and CYT-18, which specifically Ahsen et al. 1991). To uncouple translation of td pre- stabilizes the folded structure of the core (Mohr et al.

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Ribosomes as group I splicing factors

1992). These results are consistent with a structural de- removed from the message but is instead precisely fect in the pre-mRNA and not with active site inhibi- skipped during translation (Atkins et al. 1990). This ri- tion. bosome ‘‘hop’’ requires a specific structure in the Comparison of td mutations revealed that stop codons mRNA. Thus, ribosomes parallel the actions of RNA >80 nucleotides upstream of the 5Ј splice site had the binding proteins. They not only resolve misfolded struc- most deleterious effects on splicing. Strikingly, positions tures but may also stabilize certain folded conforma- −81 to −73 in the 5Ј exon are complementary to 9 bases tions, perhaps by bringing them into tRNA sites. at the 3Ј end of the intron (Fig. 1). These nucleotides The work of Semrad and Schroeder provides an ex- normally form helices within the intron (P9.0a and ample in which in vivo splicing depends on the exon P9.0b) that stabilize the catalytic core and permit 3Ј context. In td, splicing is inhibited by improper base- splice site recognition (Jaeger et al. 1993). As a result, pairing between the 5Ј exon and the 3Ј end of the intron. premature base-pairing of the intron terminus with the Self-splicing of Tetrahymena pre-rRNA is attenuated by 5Ј exon would inhibit self-splicing. Consistent with this a conserved rRNA hairpin in the 5Ј exon that competes model, splicing of pre-mRNA containing a stop codon at with the 5Ј splice site helix (Woodson and Cech 1991). −82 is increased from <1% to 60% of wild-type levels by The equilibrium between these alternative conforma- mutations in the 5Ј exon that destroy complementarity tions must be balanced to ensure splicing of the pre- with the intron (Semrad and Schroeder 1998). rRNA, and refolding of the rRNA into its conserved sec- Semrad and Schroeder propose a model in which ribo- ondary structure after the intron is removed. This bal- somes unfold the 5Ј exon RNA as they move down the ance is maintained partly by a second alternative hairpin nascent transcript, terminating at the stop codon just in the 5Ј exon that stabilizes the active form of the pre- after the 5Ј splice site (Fig. 1). As transcription continues, rRNA (Woodson and Emerick 1993) in a manner similar the intron begins to fold into the tertiary structure re- to translational attenuation (see below). In other cases, quired for self-splicing. Ribosomes upstream prevent in- exon structure promotes self-splicing. The structure of appropriate base-pairing between the 3Ј end of the intron the tRNA enhances self-splicing of group I pre- and the 5Ј exon that could compete with productive in- tRNA in Anabaena (Zaug et al. 1993). Exon secondary teractions in the intron. Splicing removes the stop at the structure has also been found to inhibit splicing of 5Ј end of the intron, permitting translation of full-length nuclear pre-mRNAs and a mitochondrial group II intron thymidylate synthase to begin. in yeast (Se´raphin et al. 1988; Libri et al. 1995). Several observations raise the question of whether An interesting question is whether the long-range pair- translation and splicing are even more tightly coordi- ing in td pre-mRNA has any regulatory function in phage nated than this model would suggest (Semrad and T4. One possibility is that this pairing is merely fortu- Schroeder 1998). It is not known whether ribosomes im- itous. It has been tolerated during T4 evolution simply mediately dissociate upon reaching the stop codon at the because upstream ribosomes normally prevent it from 5Ј end of the intron, or whether they remain temporarily interfering with splicing. The other possibility is that it associated with the pre-mRNA. However, the failure of provides an escape mechanism for expression of the mutations between −81 and −73 to restore splicing to homing endonuclease. Under normal conditions, trans- wild-type levels, and the fact that stop codons close to lation of the internal ORF is repressed by a stem–loop in the 5Ј splice site also affect splicing, lead Semrad and the td pre-mRNA (Gott et al. 1988). The ORF is ex- Schroeder to suggest that the ribosome also stabilizes the pressed from an internal late promoter within the intron; intron structure. This could occur as the result of direct the stem–loop cannot form in transcripts from the late interactions between the ribosome and the folded intron promoter (Gott et al. 1988). If T4 infects a cell in which RNA. the translational capacity is reduced, unspliced td RNA There are several reasons to think that group I introns is likely to accumulate. Perhaps under these conditions can bind to ribosomes. First, one face of the intron re- the endonuclease can be translated from the misfolded sembles the L-shape of tRNA and the td intron competes pre-mRNA, stimulating recombination among T4 genes with tRNA for binding to CYT-18 tRNA synthetase (Belfort 1990). (Caprara et al. 1996, and references therein). Second, the The idea that ribosomes alter the equilibrium between Tetrahymena group I intron becomes tightly associated alternative states of the mRNA reaches back to the clas- with 50S ribosomes when expressed in E. coli (Nikol- sic discovery of translational attenuation and antitermi- cheva and Woodson 1997) and is able to integrate into its nation of the Trp operon in E. coli (Yanofsky 1981). splice junction in 23S rRNA by a reversal of the self- When tryptophan is abundant, ribosomes read through splicing reaction (Roman and Woodson 1995). The inte- Trp codons in the 5Ј leader and prevent folding of an gration sites are concentrated in regions of the 23S rRNA antiterminator stem–loop, leading to transcription ter- that interact with tRNA and elongation factors. Prelimi- mination. In low tryptophan, the antiterminator stem nary results show that the td intron is also bound by E. prevents termination by sequestering nucleotides that coli ribosomes (R. Schroeder, pers. comm.). Finally, E. form part of the downstream terminator stem–loop. In coli 70S ribosomes can accommodate a variety of RNA Bacillus subtilis, an unusual RNA-binding protein, structures, including 10SaRNA, which has the proper- TRAP, takes the place of the ribosome (Gollnick 1994). ties of both tRNA and mRNA (Keiler et al. 1996). Within TRAP binds the RNA and disrupts the antiterminator T4, gene 60 contains an intervening sequence that is not stem in a tryptophan-dependent manner. There are now

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Woodson many other examples, such as the S4 operon in E. coli References (Spedding and Draper 1993), in which translation initia- tion is controlled by specific structures in the mRNA. Atkins, J.F., R.B. Weiss, and R.F. Gesteland. 1990. Ribosome Many examples of translational and antisense regula- gynmastics: Degree of difficulty 9.5, style 10.0. Cell 62: 413– tion in bacteria have made it clear that not only the 423. Banerjee, A.R. and D.H. Turner. 1995. The time dependence of equilibrium between two structures, but also the kinet- chemical modification reveals slow steps in the folding of a ics of RNA folding can be effectively exploited to control group I ribozyme. Biochemistry 34: 6504–6512. gene expression (e.g., Ma et al. 1994; Gultyaev et al. Belfort, M. 1989. Bacteriophage introns: Parasites within para- 1995; Poot et al. 1997). Translation of the MS2 coat pro- sites? Trends Genet. 5: 209–216. tein is repressed by a cloverleaf secondary structure in ———. 1990. 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Ironing out the kinks: splicing and translation in bacteria

Sarah A. Woodson

Genes Dev. 1998, 12:

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