Role of adenine functional groups in the recognition of the 3′-splice-site AG during the second step of pre-mRNA splicing

Abstract

The AG dinucleotide at the 3 splice sites of metazoan nuclear pre-mRNAs plays a critical role in catalytic step II of the splicing reaction. Previous studies have shown that replacement of the guanine by adenine in the AG (AG 3 GG) inhibits this step. We find that the second step was even more severely inhibited by cytosine (AG 3 CG) or uracil (AG 3 UG) substitutions at this position. By contrast, a relatively moderate inhibition was observed with a hypoxanthine substitution (AG 3 HG). When adenine was replaced by a purine base (AG 3 PG) or by 7-deazaadenine (AG 3 c 7 AG), little effect on the second step was observed, suggesting that the 6-NH 2 and N 7 groups do not play a critical role in adenine recognition. Finally, replacement of adenine by 2-aminopurine (AG 3 2-APG) had no effect on the second step. Taken together, our results suggest that the N 1 group of adenine functions as an essential determinant in adenine recognition during the second step of pre-mRNA splicing.T he accurate removal of introns from metazoan pre-mRNAs proceeds by a two-step mechanism that involves two in-line transesterification reactions. The first step of splicing is initiated by a nucleophilic attack by the 2Ј-hydroxyl group of the branchpoint adenosine on the phosphodiester bond of the guanine residue at the 5Ј end of the intron. This step results in the generation of a lariat intermediate containing a 2Ј-5Ј phosphodiester bond and the free 5Ј exon. In the second step, the 3Ј-hydroxyl group at the terminus of the 5Ј exon attacks the phosphodiester bond of the 3Ј splice site, resulting in ligation of the two exons and the concomitant release of the lariat intron (1-4).The splicing reaction takes place within the spliceosome, a 50-60S ribonucleoprotein complex consisting of five small nuclear RNAs (snRNAs), U1, U2, U4, U5, and U6, and more than 50 distinct proteins. Spliceosome assembly involves the formation of four discrete complexes on the pre-mRNA, in the order E, A, B, and C (5-7). Commitment of pre-mRNA to the splicing pathway takes place in the E (early) complex, which contains U1 small nuclear ribonucleoprotein (snRNP) as well as several non-snRNP proteins, including the essential splicing factor U2AF (8-11). The binding of U2 snRNP to the branchpoint sequence leads to the formation of complex A, whereas the subsequent entry of U4͞U6⅐U5 tri-snRNP generates complex B. Finally, a series of conformational changes that dislodge U1 and U4 snRNPs creates the catalytically active spliceosome complex C (12).In higher eukaryotes, three distinct intron sequences are required for spliceosome assembly and pre-mRNA splicing: the 5Ј splice site, the branchpoint sequence, and the 3Ј splice site, (/GURAGY, YNYURAC, and YAG/, respectively; where a slash (/) denotes a splice site; N denotes any nucleotide, R denotes purine, and Y denotes pyrimidine; underlining indicates the conserved nucleotides). Genetic and biochemical analyses suggest that a number of RNA-RNA interactions involving snRNAs and these sequences are responsible for the select...

Aberrant 3′ splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization

“…However, comparison of the observed and expected distributions derived from di-nucleotide mutability rates that allow for the influence of neighbouring nucleotides ( 48 ) failed to confirm any bias for both intron positions ( Table 3 ). Thus, although small effects of ‘leaky’ dinucleotides on the observed distribution cannot be excluded, these data are consistent with dramatic consequences for splicing of any point mutation in the highly conserved 3′AG and with indistinguishable defects of the second splicing step previously observed in vitro both for intron position −1 ( 51 ) and −2 ( 52 ).…”

Crystal structure of an RNA duplex containing phenyl-ribonucleotides, hydrophobic isosteres of the natural pyrimidines

“…The crystal structure of an RNA duplex with incorporated phenyl ribonucleotides determined at a resolution of 1+97 Å reveals that the phenyl analog can stack between bases, resulting only in minor deviations from a standard A-form geometry of the duplex+ In the crystallized sequence, phenyls are arranged opposite each other forming a pseudo-base pair with van der Waals contacts between phenyl rings across strands+ The preferred pairing between hydrophobic base analogs observed here is consistent with earlier results in DNA that demonstrated self-pairing between hydrophobic isosteres in DNA duplexes (Schweitzer & Kool, 1995)+ The structural results suggest that phenyl may be a better mimetic of the natural pyrimidines than one might have expected for a base analog lacking any hydrogen bond donors or acceptors+ This is in line with the wild-type activities of hammerhead ribozymes bearing phenyl residues at key sites and the observation that a premRNA substrate with a phenyl instead of A at the 39 splice site is a better substrate than those carrying a G, C, or U substitution (Gaur et al+, 2000)+ Matching shape, enhanced conformational flexibility, and alternative stacking interactions, involving dipolar and quadrupolar effects, may allow the phenyl analog to overcome the absence of hydrogen-bonding functions and to mimic the interactions of wild-type bases at the active site of ribozymes and spliceosomes+…”

“…A: Preparation of 1-Deoxy-1-phenyl-b-D-ribofuranose derivative suitable for oligonucleotide synthesis; compound 5 corresponds to the phenyl ribonucleoside+ Reagents: i: Triflic acid, benzyl 2,2,2-trichloroacetimidate; ii: Phenyllithium; Ϫ78 8C; iii: Triethylsilane, boron trifluoride etherate, Ϫ40 8C; iv: Boron tribromide, Ϫ78 8C; v: 4,49-Dimethoxytrityl chloride, pyridine; vi: Silver nitrate, t-butyldimethylsilyl chloride, tetrahydrofuran/pyridine; vii: 5% Triethylamine in methanol; viii: 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite, N-methylimidazole, N,N-diisopropylethylamine+ B: Possible structures for single-and double-stranded arrangements of the RNA octamer CCCPGGGG+ Cytosines are drawn as open boxes, guanines are dashed, and phenyls are black+ From left to right: duplex with two P-G mismatches; hairpin with three C-G base pairs in the stem and a P-G loop; duplex with a central purine-purine base pair and loopedout Ps; duplex with strands slid along each other by one base-pair step, generating a central P-P pair and 3-terminal G overhangs+ The arrangement on the far right is observed in the crystal structure+ 9 with phenyl decreased the catalytic rate only 20-fold compared to the 2,000-fold deleterious effect resulting from removal of the adenine base+ Another surprising observation was the almost wild-type activity of a nuclear pre-mRNA variant with an adenine r phenyl mutation at the 39-splice AG during the second step of the splicing reaction (Gaur et al+, 2000)+ The consequences of hydrophobic, non-hydrogenbonding bases and base pairs for duplex stability and nucleic acid-protein interactions have been investigated in some detail with DNA but remain largely unexplored in the case of RNA+ Thus, difluorotoluene (F), a nonpolar analog of thymine codes specifically for adenine replication (Moran et al+, 1997) and the geometry of the F-A pair closely resembles that of T-A, although F causes destabilization of the DNA duplex (Guckian et al+, 1998)+ Similarly, a pyrene nucleoside analog shows significant selectivity for a model abasic site over the natural bases (Matray & Kool, 1998) and polymerases efficiently incorporate the pyrene residue opposite a template site lacking a base (Matray & Kool, 1999)+ In DNA duplexes, nonpolar isosteres of adenine and thymine pair with a stability that is similar to that of the T-G mismatch base pair (Schweitzer & Kool, 1995)+ Moreover, at the ends of helices such hydrophobic pairs can be more stabilizing than a canonical A-T pair and, interestingly, hydrophobic analogs prefer to pair with a hydrophobic partner rather than a natural base+ Attached at the 59-terminus of a DNA duplex as single dangling nucleotides, nonpolar aromatic analogs were found to be equally or more stabilizing than the four natural bases (Guckian et al+, 2000)+ Here, we report a new chemical synthetic route for preparing the phenyl ribonucleotide+ To shed light on the energetic and structural consequences of the incorporation of the phenyl nucleotide analog into an RNA duplex, we analyzed the thermodynamic stability of RNA octamers with single phenyl residues (P) and determined the crystal structure of r(CCCPGGGG)+ The structure gives the first detailed picture of the interactions of the hydrophobic phenyl nucleotide in the context of an RNA duplex and may aid in the rationalization of the accumulated activity data for RNAs bearing the phenyl modification+…”

A mutational analysis of U12-dependent splice site dinucleotides

“…In the case of U2-dependent introns, previous investigations of the effects of mutations in the U of the 5 0 /GU and the A residue of the 3 0 AG/ dinucleotides showed that these residues were important for efficient splicing in vivo and in vitro (Reed and Maniatis 1985;Aebi et al 1986;Ruis et al 1994;Gaur et al 2000). Unlike the case of the terminal G residues, no combinations of 5 0 and 3 0 mutations of the U and A residues restored function (Ruis et al 1994).…”

“…The only notable exception is the low frequency occurrence of U2-dependent 5 0 splice sites with a +2 C residue. Most mutations of these positions have been shown to be highly defective for splicing in U2-dependent introns (Reed and Maniatis 1985;Aebi et al 1986;Ruis et al 1994;Gaur et al 2000). We have shown previously that mutation of the 3 0 splice site À2 A residue in a U12-dependent intron blocks splicing in vivo (Dietrich et al 1997(Dietrich et al , 2001).…”