[29] Analysis of ultraviolet-induced RNA-RNA cross-links: A means for probing RNA structure-function relationships

Branch, Andrea D.; Benenfeld, Bonnie J.; Paul, Cynthia P.; Robertson, Hugh D.

doi:10.1016/0076-6879(89)80115-6

Cited by 33 publications

(22 citation statements)

References 16 publications

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“…Moreover, these findings are consistent with the suggestion that UV cross-linking is favored between bases that are in close proximity but not involved in Watson-Crick interactions (Branch et al 1989;Mitchell et al 1990). 5'SS RNA oligos with mutations at positions + 1, + 3, + 4, or + 5 cross-linked to U1 snRNA with an efficiency that was reduced 2-3-fold as compared to wild type ( Fig.…”

Section: Effects Of Single Point Mutations In the 5'ss On Interactionsupporting

confidence: 78%

U4/U5/U6 snRNP recognizes the 5' splice site in the absence of U2 snRNP.

Konforti

Konarska²

1994

Genes Dev.

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Using an in vitro system in which a 5' splice site (5'SS) RNA oligo (A_AG $ GUAAGUAdT) is capable of inducing formation of U2/U4/U5/U6 snRNP complex we show that this oligo specifically binds to U4/U5/U6 snRNP and cross-links to U6 snRNA in the absence of U2 snRNP. Moreover, 5'SS RNA oligo bound to U4/U5/U6 snRNP is chased to U2/U4/U5/U6 snRNP complex upon addition of U2 snRNP. Recognition of the 5'SS by U4/U5/U6 snRNP correlates with the 5'SS consensus sequence. Unlike the interaction with U1 snRNP, this recognition depends largely on interactions other than RNA-RNA base pairing. Finally, the region of U6 snRNA required for this interaction with U4/U5/U6 snRNP is positioned upstream of stem I in the U4-U6 structure. We propose that the 5'SS-U4/US/U6 snRNP complex is an intermediate in spliceosome assembly and that recognition of the 5'SS by U4/U5/U6 snRNP occurs after the 5'SS-U1 snRNA base pairing is disrupted but before the U4-U6 snRNA structure is destabilized.[Key Words: 5' splice site recognition; spliceosome assembly~ snRNPs] Received May 13, 1994~ revised version accepted July 1, 1994.Removal of introns from nuclear precursor messenger RNA {pre-mRNA} is mediated by a large multicomponent complex called the spliceosome which is composed of U1, U2, U4, U5, and U6 small nuclear ribonucleoprotein particles (snRNPs} and numerous proteins (for review, see Green 1991; Guthrie 1991; Moore et al. 1993).In mammals, spliceosome assembly depends on a consensus sequence at the 5' splice site (5'SS}, a branch site with an adjacent polypyrimidine tract, and a 3'SS consensus sequence. Initially, U 1 snRNP binds via base pairing with the 5'SS and commits the pre-mRNA to the splicing pathway. Subsequently, U2 snRNP binds at the branch site to form splicing complex A, which is converted into splicing complex B upon association of U4/ U5/U6 triple snRNP. Within U4/U5/U6 snRNP, U4, and U6 snRNAs are extensively base paired. Prior to {or concomitant with} the first step of splicing, this U4-U6 base pairing is disrupted and U4 snRNP is released. Because the pairing interaction between U6 and U4 snRNAs has been proposed to negatively regulate U6 and is mutually exclusive with the pairing interaction between U6 and U2 snRNAs, this structural rearrangement could represent the catalytic activation of the spliceosome.

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Section: Effects Of Single Point Mutations In the 5'ss On Interactionsupporting

confidence: 78%

U4/U5/U6 snRNP recognizes the 5' splice site in the absence of U2 snRNP.

Konforti

Konarska²

1994

Genes Dev.

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“…1) was used to seek a UV-crosslinkable element of local tertiary structure in the HDV antigenomic strand. After UV treatment as before (Branch et al 1989a(Branch et al , 1989c, two-dimensional polyacrylamide gel analysis revealed a UV-dependent species that ran with a distinctively lower mobility in the second-dimension denaturing gel. Partial RNase T1 digestion and fingerprinting (Branch et al 1989b(Branch et al , 1989c) showed a single, new oligonucleotide among the specific antigenomic HDV sequences that had undergone UV crosslinking.…”

Section: Antigenomic Hdv Has a Tertiary Structural Element In The Conmentioning

confidence: 99%

“…After UV treatment as before (Branch et al 1989a(Branch et al , 1989c, two-dimensional polyacrylamide gel analysis revealed a UV-dependent species that ran with a distinctively lower mobility in the second-dimension denaturing gel. Partial RNase T1 digestion and fingerprinting (Branch et al 1989b(Branch et al , 1989c) showed a single, new oligonucleotide among the specific antigenomic HDV sequences that had undergone UV crosslinking. Sequences were determined by direct means (Barrell 1971;Branch et al 1989b), with the exact location of the crosslink (linking U701-U889) confirmed by primer extension analysis as before (Lyons et al 2001).…”

Section: Antigenomic Hdv Has a Tertiary Structural Element In The Conmentioning

confidence: 99%

“…Our laboratory has worked out techniques for producing specific UV crosslinks in RNA molecules at sites of preexisting local tertiary structure (Branch et al 1985a(Branch et al , 1989c, isolating the RNAs containing them by altered behavior on polyacrylamide gels, and their specific regions by partial RNase T1 digestion (Branch et al 1985b(Branch et al , 1989c, and mapping their exact positions in the RNA sequence (Branch et al 1989a(Branch et al , 1989b(Branch et al , 1989c(Branch et al , 1990aLyons et al 2001). For antigenomic HDV, a transcript containing the conserved domain, spanning residues 482-963 (shown in Fig.…”

Section: Antigenomic Hdv Has a Tertiary Structural Element In The Conmentioning

confidence: 99%

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Recurring features of local tertiary structural elements in RNA molecules exemplified by hepatitis D virus RNA

Circle

Lyons²,

Neel³

et al. 2003

RNA

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Elements of local tertiary structure in RNA molecules are important in understanding structure-function relationships. The loop E motif, first identified in several eukaryotic RNAs at functional sites which share an exceptional propensity for UV crosslinking between specific bases, was subsequently shown to have a characteristic tertiary structure. Common sequences and secondary structures have allowed other examples of the E-loop motif to be recognized in a number of RNAs at sites of protein binding or other biological function. We would like to know if more elements of local tertiary structure, in addition to the E-loop, can be identified by such common features. The highly structured circular RNA genome of the hepatitis D virus (HDV) provides an ideal test molecule because it has extensive internal structure, a UV-crosslinkable tertiary element, and specific sites for functional interactions with proteins including host PKR. We have now found a UV-crosslinkable element of local tertiary structure in antigenomic HDV RNA which, although differing from the E-loop, has a very similar pattern of sequence and secondary structure to the UV-crosslinkable element found in the genomic strand. Despite the fact that the two structures map close to one another, the sequences comprising them are not the templates for each other. Instead, the template regions for each element are additional sites for potential higher order structure on their respective complementary strands. This wealth of recurring sequences interspersed with base-paired stems provides a context to examine other RNA species for such features and their correlations with biological function.

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“…Following UV cross-linking this site is so resistant to cleavage by RNase T1 that 16 of its constituent G residues are not cleaved by 1 mg/ml RNase Ti during a 45 min incubation at 37°C in 10 mM Tris, 1 mM EDTA (29,33). While UV-induced cross-links often inhibit nuclease cleavage at bonds directly neighboring cross-linked bases (35), the extensive nuclease resistance of the UV crossa b c d e f g hi 2_* Figure 2. RNA-1 in the RNA-l-RNA-2 complex is not cleaved by a delta ribozyme, RNA-3.…”

Section: Introductionmentioning

confidence: 99%

T_mstudies of a tertiary structure from the human hepatitis delta agent which functionsin vitroas a ribozyme control element

1995

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Viroids and other circular subviral RNA pathogens, such as the hepatitis delta agent, use a rolling circle replication cycle requiring an intact circular RNA. However, many infectious RNAs have the potential to form self-cleavage structures, whose formation must be controlled in order to preserve the circular replication template. The native structure of delta RNA contains a highly conserved element of local tertiary structure which is composed of sequences partially overlapping those needed to form the self-cleavage motif. A bimolecular complex containing the tertiary structure can be made. We show that when it is part of this bimolecular complex the potential cleavage site is protected and is not cleaved by the delta ribozyme, demonstrating that the element of local tertiary structure can function as a ribozyme control element in vitro. Physical studies of the complex containing this element were carried out. The complex binds magnesium ions and is not readily dissociated by EDTA under the conditions tested; >50%/o of the complexes remain following incubation in 1 mM EDTA at 600C for 81 min. The thermal stability of the complex is reduced in the presence of sodium ions. A DNA complex and a perfect RNA duplex studied in parallel showed a similar effect, but of lesser magnitude. The RNA complex melts at temperatures -10°C lower in buffers containing 0.5 mM MgCI2 and 100 mM NaCI than in buffers containing 0.5 mM MgCI2 with no NaCI (78.1 compared with 87.70C). The element of local tertiary structure in delta genomic RNA appears to be a molecular clamp whose stability is highly sensitive to ion concentration in the physiological range.

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[29] Analysis of ultraviolet-induced RNA-RNA cross-links: A means for probing RNA structure-function relationships

Cited by 33 publications

References 16 publications

U4/U5/U6 snRNP recognizes the 5' splice site in the absence of U2 snRNP.

U4/U5/U6 snRNP recognizes the 5' splice site in the absence of U2 snRNP.

Recurring features of local tertiary structural elements in RNA molecules exemplified by hepatitis D virus RNA

T_mstudies of a tertiary structure from the human hepatitis delta agent which functionsin vitroas a ribozyme control element

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[29] Analysis of ultraviolet-induced RNA-RNA cross-links: A means for probing RNA structure-function relationships

Cited by 33 publications

References 16 publications

U4/U5/U6 snRNP recognizes the 5' splice site in the absence of U2 snRNP.

U4/U5/U6 snRNP recognizes the 5' splice site in the absence of U2 snRNP.

Recurring features of local tertiary structural elements in RNA molecules exemplified by hepatitis D virus RNA

Tmstudies of a tertiary structure from the human hepatitis delta agent which functionsin vitroas a ribozyme control element

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T_mstudies of a tertiary structure from the human hepatitis delta agent which functionsin vitroas a ribozyme control element