"Poly(A) Tail Recognition by a Viral RNA Element Through Assembly of a Triple Helix".

Rachel M. Mitton-Fry ¹, Suzanne J. DeGregorio ¹, Jimin Wang ², Thomas A. Steitz ^{2, 3, 4}, and Joan A. Steitz ¹,*

¹ Department of Molecular Biophysics and Biochemistry (MB&B), Howard Hughes Medical Institute (HHMI), Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536–9812, USA.
² Department of MB&B, Yale University, Bass Center for Molecular and Structural Biology, 266 Whitney Avenue, New Haven, CT 06520–8114, USA.
³ Department of Chemistry, Yale University, New Haven, CT 06520–8107, USA.
⁴ HHMI, Yale University, Bass Center for Molecular and Structural Biology, 266 Whitney Avenue, New Haven, CT 06520–8114, USA.

*To whom correspondence should be addressed. E-mail: joan.steitz@yale.edu

Received for publication 30 July 2010.   Accepted for publication 15 October 2010.

Kaposi’s sarcoma–associated herpesvirus produces a highly abundant, nuclear noncoding RNA, polyadenylated nuclear (PAN) RNA, which contains an element that prevents its decay. The 79-nucleotide expression and nuclear retention element (ENE) was proposed to adopt a secondary structure like that of a box H/ACA small nucleolar RNA (snoRNA), with a U-rich internal loop that hybridizes to and protects the PAN RNA poly(A) tail. The crystal structure of a complex between the 40-nucleotide ENE core and oligo(A)₉ RNA at 2.5 angstrom resolution reveals that unlike snoRNAs, the U-rich loop of the ENE engages its target through formation of a major-groove triple helix. A-minor interactions extend the binding interface. Deadenylation assays confirm the functional importance of the triple helix. Thus, the ENE acts as an intramolecular RNA clamp, sequestering the PAN poly(A) tail and preventing the initiation of RNA decay.

Kaposi’s sarcoma–associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma (KS), the most common AIDS-associated cancer (1, 2). Although largely controlled in the developed world by antiretroviral treatment against HIV, KS has become one of the most prevalent cancers in Africa (3). KSHV is a g-herpesvirus that exists in either a latent or lytic state. During the lytic phase, KSHV produces PAN (polyadenylated nuclear) RNA, a 1.1-kb noncoding RNA with a 5' cap and 3' poly(A) tail (4–6) that is retained in the nucleus of infected cells. Although the function of PAN RNA is not known, it accumulates to extremely high levels, amounting to as much as 80% of the polyadenylated RNA in the cell (4, 6). The expression and nuclear retention element (ENE), a 79-nucleotide (nt) element located near the 3' end of PAN RNA, is responsible for this accumulation (7). The ENE prevents deadenylation and decay of PAN RNA through direct, cis-acting sequestration of the poly(A) tail (8). The ENE also abrogates rapid nuclear decay when inserted into polyadenylated mRNAs lacking introns (8), apparently through protection from deadenylation, the first step in degradation of eukaryotic mRNAs (9, 10). Secondary structure prediction and analyses of mutants using cellular assays suggested that the ENE forms a hairpin containing a U-rich internal loop flanked by short helices, reminiscent of box H/ACA small nucleolar RNA (snoRNA) hairpins (Fig. 1A) (8, 11). The internal loops of box H/ACA snoRNAs base pair with target regions in ribosomal RNA (rRNA) to direct the conversion of specific uridine residues to pseudouridine (12). By analogy, the ENE internal loop was predicted to base pair with the poly(A) tail of PAN RNA (Fig. 1A), protecting it from exonucleolytic digestion (11).

Fig. 1: Structural overview of the complex formed between the ENE core and A₉ RNA.

Fig. 1: Structural overview of the complex formed between the ENE core and A₉ RNA.

(A) Schematic diagram of PAN RNA, showing the interaction between the ENE and the poly(A) tail that was previously proposed (8, 11), with the ENE core in green. For crystallization, the upper stem of this central sequence was capped with a C-G base pair and a GAAA tetraloop and the lower stem was followed by a 3' C nucleotide (to produce a blunt-ended helix) (Fig. 1D).

(B) Ribbon representation of the crystal structure, with the ENE core in green, the non-native sequence in gray, and the A₉ oligonucleotide in magenta. Four copies of the ENE complex are found in the asymmetric unit of the crystal, two pairs of complexes related by noncrystallographic symmetry (NCS). The two non-NCS–related complexes are almost identical in the central region [RMSD = 0.57 Å over all nonhydrogen atoms in the triple helix (fig. S3)].

(C) 90° rotation of the image shown in (B).

(D) Schematic diagram of the complex [colors as in (B and C)], with Leontis-Westhof notation (26) indicating major-groove triple helix contacts and dotted lines indicating A-minor interactions. The base of the bulged nucleotide A32 (outlined) is disordered.

To elucidate the mode of interaction between the ENE and the poly(A) tail of PAN RNA, we have determined the crystal structure of a complex formed between the 40-nt ENE core and oligo(A) RNA and studied it biochemically (13). Electrophoretic mobility shift assays showed that the isolated ENE is capable of binding oligo(A) in trans (fig. S1). U to C mutations in the internal loop greatly decrease the interaction between the ENE and oligo(A), whereas truncations of the ENE that retain the helix-loop-helix core (Fig. 1A) have no effect (fig. S1). These results are consistent with those obtained from in vivo and in vitro assays of ENE mutants (11). Nuclear magnetic resonance (NMR) studies confirmed that the ENE core alone adopts a helix-loop-helix conformation (fig. S2A). Increased spectral complexity in the imino region of one-dimensional 1H spectra upon A10 addition suggested that the loop becomes involved in hydrogen-bonding interactions with the oligo(A) (fig. S2B). Crystallization trials were then performed using several ENE core sequences that varied in the helix length mixed with a variety of short A-rich RNA oligonucleotides. Optimal crystallization occurred with the 40-nt ENE core construct and A9 RNA. Data from crystals soaked in buffer solutions containing iridium hexamine were used to determine the phases, and the final structure was refined to 2.5 Å resolution (<I/s₁> = 1.0; <I/s₁> = 2.0 at 2.65 Å resolution) with working and free R factors (Rwork and R/Rfree) of 21.8%/23.3%, respectively (figs. S3 to S5 and table S1).

The crystal structure reveals that the ENE core forms a triple-stranded complex with its bound A₉ oligonucleotide (Fig. 1). The ENE assumes the expected secondary structure, with Watson-Crick stems flanking a U-rich internal loop (10). Instead of forming base pairs around this loop, however, the A₉ RNA adopts an extended conformation and interacts with both the ENE loop and the lower stem. Nucleotides A5 to A9 of the A₉ oligonucleotide simultaneously engage both sides of the internal loop in an extended U-A•U major-groove triple helix (Fig. 2A). Here, the five consecutive A nucleotides form Watson-Crick base pairs with the five consecutive U nucleotides on the 3' side of the loop [U27 to U31 (14)]. The 5' side of the ENE internal loop lies in the major groove of the resulting helix, with the Watson-Crick face of nucleotides U8 to U12 base pairing with the Hoogsteen face of the A nucleotides (Fig. 2B). Hydrogen bonds are also observed between the A₉ phosphate backbone and the ribose hydroxyl groups of the Hoogsteen U strand. The base triples formed are nearly planar, and there is no direct contact between the two U strands of the internal loop (Fig. 2, A and B). The helical axis of the complex is nearly straight through the transitions between the triple helix and the flanking ENE stems. The additional two nucleotides of the 3' side of the ENE internal loop (A32 and U33) bulge out, allowing continuous stacking between the lower stem and the A and Hoogsteen strands of the triple helix. Despite the presence of the Hoogsteen strand in the A:U major groove, a deep groove remains (fig S6).

Fig. 2: Detailed views of key structural interactions between the ENE and A₉ RNA.

Fig. 2: Detailed views of key structural interactions between the ENE and A₉ RNA.

(A) Close-up of the major-groove triple helix formed by 5 nt of A₉ and the internal U-rich loop of the ENE hairpin (colors as in Fig. 1, with the bases involved in Watson-Crick pairing in yellow, hydrogen bonds within base triples in cyan, and the A₉ nucleotides labeled in italics). The U strand that makes Hoogsteen interactions in the major groove is shown in the foreground.

(B) Superposition of the five ENE:A₉ U-A•U base triples showing the regularity of the triple-helical structure.

(C) Close-up of the A-minor triad, with Watson-Crick base pairing and hydrogen bonds involving the A strand in cyan.

(D) A-minor interactions with the lower ENE stem. Crystal-packing interactions involving nucleotide A1 (fig. S3A) most likely pull the N2 of nucleotide A2 just beyond hydrogen-bonding distance from the 2' OH of C36 in the Type III interaction.

The binding interface is augmented by a triad of A-minor interactions formed between the bound A9 oligonucleotide and the lower stem of the ENE. Nucleotides A2 to A4 contact the three consecutive G-C base pairs that close the lower stem (Fig. 2C). As previously described for 23S rRNA in the high-resolution structure of the 50S ribosome (15), the three As of the triad penetrate with increasing depth into the G:C minor groove from the 5' to 3' direction, such that the initial interaction is a type III, the next is a type II, and the last is a type I A-minor interaction (Fig. 2D) (15, 16).

The interactions made between the ENE core and A₉ RNA are strikingly different from those determined for the box H/ACA snoRNAs and their target rRNA sequences (Fig. 3, A and B). In the snoRNA structures, the substrate strand is sharply kinked, forming parallel Watson-Crick helices with the two strands of the snoRNA internal loop, which then stack coaxially on the flanking snoRNA hairpin helices (17, 18). The crystal structure described here involves an intermolecular interaction between the core ENE and A₉ RNA. However, as the ENE has only been observed to protect the PAN RNA poly(A) tail in an intramolecular manner (8), these elements formally adopt a pseudoknot structure in the context of the full-length RNA (see supporting online material). Consistent with this, the ENE:A₉ complex resembles H-type pseudoknots observed in telomerase RNA (Fig. 3, C and D) (19) and the S-adenosylmethionine-responsive riboswitch SAM-II (fig. S7) (20). Although the topology of the surrounding stems is different in the H-type pseudoknots, the structures of the major-groove triple helices can be superimposed with that of the ENE complex with root mean square deviations (RMSDs) of ~1 Å. Felsenfeld, Davies, and Rich first studied RNA triple helices composed of poly(U)-poly(A)•poly(U) more than half a century ago (21, 22). Denaturation studies later suggested that DNA hairpins with T-rich loops likewise bind oligo(dA) through formation of T-A•T triple helices (23). Analogous interactions between RNA hairpins and A-rich sequences were also proposed (23). However, the ENE is the first natural example of the use of a U-rich internal loop to capture and sequester a poly(A) RNA sequence.

Fig. 3: Comparison of the ENE:A₉ complex with other structures.

Fig. 3: Comparison of the ENE:A₉ complex with other structures.

(A) The complex between the ENE and A₉ RNA (top three base triples boxed).

(B) The solution structure of a complex between a truncated H/ACA snoRNA and a 14-nt sequence corresponding to its rRNA substrate [PDB 2P89, model 1 (18)]. The snoRNA hairpin is shown in green, with the bound substrate in yellow.

(C) The solution structure of the human telomerase pseudoknot [PDB 2K95, model 1 (27)]. The triple helix (boxed) is composed of three U-A•U base triples, and the strand contributing these A nucleotides is shown in cyan, whereas the strands that contribute the U nucleotides are shown in brown.

(D) Superposition of the triple helix from the ENE:A₉ complex with the triple helix from the telomerase RNA pseudoknot. Colors and orientations are as in (A) and (C). The top three U-A•U base triples (involving A7 to A9) from the ENE:A₉ complex were used for superposition (RMSD = 1.1 Å over all nonhydrogen atoms).

We used deadenylation assays to confirm the functional importance of the ENE:A₉ triple helix described here (13). Mutation of a single U to C in either side of the ENE U-rich pocket had previously been shown to decrease protection of PAN RNA’s poly(A) tail from deadenylation in nuclear extract (11). Simultaneous U to C mutations (mutating one nucleotide from each side of the pocket) were as deleterious as deletion of the entire ENE. Because the two U nucleotides mutated in that experiment contact the same A in the crystal structure (Fig. 4A), we postulated that the loss of ENE function arising from disruption of a single U-A•U base triple might be restored by replacement with a nearly isosteric C-G•C base triple (Fig. 4B) (24). Native gel shift analysis supported this proposal, as ENEs containing simultaneous U to C mutations contacting the same A in the crystal structure can bind an oligo(A) molecule containing a single A to G substitution (A₇GA₂) (fig. S8, A and C), whereas ENE constructs with mutations to C in U residues that contact two different A nucleotides in the crystal structure cannot (fig. S8, B and C).

Fig. 4: Triple-helix assembly protects the PAN RNA poly(A) tail from deadenylation.

Fig. 4: Triple-helix assembly protects the PAN RNA poly(A) tail from deadenylation.

(A) Cartoon of the ENE:A₉ complex structure in the context of full-length PAN, highlighting the locations of U903 and U949 (numbering from the PAN RNA 5' end).

(B) Comparison of U-A•U and C-G•C+ base triples [colors as in (A), with the hydrogen bond formed upon protonation of the Hoogsteen C nucleotide in blue].

(C) In vitro deadenylation assays show that single G substitutions in the poly(A) tail rescue a nonfunctional double-mutant ENE by formation of C-G•C base triples (24). The substrates consist of the 327-nt 3' terminus of PAN RNA followed by a 60-nt tail either composed of all adenylate (A₆₀) or with single G substitutions 3 or 41 nucleotides from the 3' end of the poly(A) tail (A₅₇GA₂ or A₁₉GA₄₀, respectively); tail identity is designated above the panels. After incubation in HeLa cell nuclear extract (28) for the indicated times, products were separated by denaturing gel electrophoresis. RNAs containing the wild-type ENE, double-mutant (U903C, U949C) ENE, and D ENE are shown in the upper, middle, and lower panels, respectively. +dT lanes refer to transcripts in which the poly(A) tail was removed by endogenous ribonuclease H after addition of oligo(dT)₄₀ to the reaction mix. The A₆₀ and A₀ labels on the left show the migration of fully adenylated and deadenylated substrates.

We thus transcribed PAN RNA deadenylation substrates that contained either wild-type or double-mutant ENE or that lacked the ENE altogether (see legend to Fig. 4C). Each of the substrates terminated in a 60-nt poly(A) tail with or without a single A to G substitution (A₆₀ or A₅₇GA₂). We assessed these constructs for ENE-dependent protection from deadenylation in nuclear extract (Fig. 4C, left and middle panels). As expected, the wild-type ENE protected the A₆₀ tail from deadenylation, whereas the A₆₀ tails of constructs containing no ENE (D ENE) or the double-mutant ENE (U903C, U949C) were not protected from deadenylation (Fig. 4C, left panels) (10). In contrast, the double-mutant ENE effectively protected the A₅₇GA₂ poly(A) tail, which has a single A to G substitution close to its 3' end (Fig. 4C, center panel). The presence of a G residue in the poly(A) tail does not on its own confer resistance to deadenylation, as the A₅₇GA₂ poly(A) tail of a construct lacking the ENE was not protected (Fig. 4C, bottom middle panel). These results, supported by the native gel shift data described above (fig. S8), indicate that the triple helix observed in the structure is critical to ENE function.

Finally, we tested the ability of the double-mutant ENE to protect a poly(A) tail containing a more internal G substitution (A19GA40) (Fig. 4C, right panel). In nuclear extract, this poly(A) tail was rapidly deadenylated to a size consistent with formation of a triple helix that includes the predicted C-G•C base triple (Fig. 4C, right middle panel). The ability of the double-mutant ENE to locate a single G within a long stretch of A nucleotides is striking. The deadenylation data also demonstrate that the ENE does not require the 3' terminus of the poly(A) tail for binding, despite the involvement of the 3' end of the A9 RNA in the final base triple of the ENE core:A₉ structure. The results further argue that no specific register is required for the interaction of the wild-type ENE with the PAN RNA poly(A) tail. The presence of multiple binding sites for the ENE along the poly(A) sequence may contribute to its ability to protect tails of various lengths from deadenylation by cellular exonucleases (Fig. 4A). How the ENE may collaborate with poly(A)–binding proteins that are known to coat the poly(A) tails of RNA polymerase II transcripts in vivo (25) remains to be determined.

The key feature of the core ENE:A9 crystal structure is a functionally important U-A•U major-groove triple helix, which is extended by A-minor interactions. The structure reveals an intramolecular clamp mechanism for recognition of poly(A) RNA and suggests how the ENE sequesters the PAN poly(A) tail from degradation by cellular deadenylases. Since viruses routinely borrow strategies from their hosts, we predict that similar mechanisms may protect some cellular noncoding RNAs from rapid turnover.

http://www.sciencemag.org/content/suppl/2010/11/22/330.6008.1244.DC1

Materials and Methods
SOM Text
Figs. S1 to S8
Table S1
References

X-ray data were collected at the National Synchrotron Light Source (X29A) at Brookhaven National Laboratory (13). Financial support for this research was provided by NIH grant CA16038 to J.A.S. and NIH grant GM022778 to T.A.S. The content is solely the responsibility of the authors and does not necessarily represent the official views of NIH.

R.M.M.-F. is supported by a Jane Coffin Childs Memorial Fund Postdoctoral fellowship. J.A.S. and T.A.S. are investigators of the Howard Hughes Medical Institute.

Coordinates and structure factors have been deposited in the Protein Data Bank under accession code 3P22.

Supporting Online Material:

Materials and Methods:

RNA preparation

Oligo(A) RNAs were purchased from Dharmacon, deprotected and dried per the
manufacturer’s instructions, and dissolved in ddH2O or crystallization buffer (below).
For gel shift assays, NMR spectroscopy, and initial crystallography screens, ENE RNAs
were transcribed from an annealed dsDNA template bearing two 2´-O-methyl nucleotides
at the 5´ end of the template strand to reduce non-templated addition of nucleotides at the
3´ end of the transcript (1). Transcription reactions (5–15 mL) included 40 mM Tris-
HCl, pH 7.5, 0.05% Triton X-100, 30 mM MgCl₂, 1.6 mM spermidine, 4 mM ATP, CTP,
and GTP, 6 mM UTP, 10 mM DTT, and 0.5 mM annealed template. Reactions used
either wild-type T7 RNA polymerase or a mutant T7 polymerase that produces fewer
abortive transcripts [S266P (2)]. After 1.5 hours at 37°C, reactions were quenched by
addition of EDTA and extracted with acidic phenol. RNAs were purified by denaturing
gel electrophoresis, followed by passive elution of the excised, crushed gel bands
overnight at 4°C. After ethanol precipitation, RNAs were exchanged into crystallization
buffer (50 mM sodium cacodylate, pH 6.5, 50 mM KCl, 1 mM MgCl₂, and 0.1 mM
EDTA).

Use of the modified template greatly reduced addition of nucleotides to the 3´ end
of the transcript. However, difficulty was encountered in separating a 39-nt (n-1)
transcript from the full-length 40-nt product. Therefore, for final crystals, we cloned the
ENE core sequence into plasmid pHDV [gift from Graeme Conn (3)], which placed the
HDV ribozyme at the 3´ end of the ENE. XbaI-linearized template (0.07 mg/mL) was
used to transcribe the ENE core sequence, which was purified as above. Ribozyme
cleavage occurred during the course of the transcription reaction. The resultant 2´,3´-
cyclic phosphate proved deleterious to ENE crystallization and was removed through
incubation of 1 mg/ml RNA with PNK (0.5 units/mL) in 50 mM Tris-HCl, pH 7.5, 10
mM MgCl₂, 10 mM DTT, 1 mM ATP, and 0.25 mg/ml BSA for 5 hours at 37°C (4).
Phosphate removal caused altered gel mobility, allowing assessment of the progress of
the reaction by ethidium bromide staining of a denaturing gel. Dephosphorylated ENE
RNA was then exchanged into ddH2O, dried under vacuum, and resuspended in
crystallization buffer (above).

Crystallization

ENE RNA (~ 5 mg/ml) was mixed with an equimolar amount of AN
oligonucleotide in crystallization buffer (above), heated to 95°C for 3 minutes, then snapcooled
on ice. Crystals were grown at 20°C by the sitting drop vapor diffusion method
using equal volumes of the RNA solution and the reservoir solution (90 mM magnesium
acetate, 17% methyl-2,4-pentanediol (MPD), and 50 mM MES, pH 5.6). After
optimization, crystals with needle-blade morphology grew over the course of several
weeks to a maximum size of ~1.5 mm x 200 mm x 20 mm. Crystals were rinsed in
reservoir solution, stabilized by addition of MPD to 50%, and then flash frozen in liquid
nitrogen. For heavy-atom derivatives, crystals were stabilized as above, soaked in
stabilization solution containing 2 mM iridium (III) hexamine trichloride (gift from Scott
Strobel) for 1–2 hours, and then flash frozen in liquid nitrogen.

Data acquisition and processing

Diffraction datasets (native and heavy atom) were acquired at the National
Synchrotron Light Source (NSLS), beamline X29A, at Brookhaven National Laboratory.
Use of the NSLS at Brookhaven was supported by the U.S. Department of Energy, Office
of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886.
For crystals soaked in iridium hexamine, datasets were collected at peak, inflection, and
high remote wavelengths. Data were indexed, integrated, and scaled in HKL2000 (5).
Crystals belong to the C2 space group with cell dimensions a=144.7 Å, b=50.9 Å, c=91.4
Å, and b=125.1°. We occasionally observed some deviations in the C-centering
symmetry of crystals, resulting in non-zero intensity for h+k=2n+1 reflections and
reducing the space group to P21. The deviations were insignificant for the crystals used
for structure determination. The crystals had a Matthews coefficient of 2.16 and a
solvent content of 62%, with four ENE:A9 complexes in the asymmetric unit.
Heavy atom datasets were nearly isomorphous with native sets. The initial
locations of the heavy atom sites were found using the program SHELXE (6) and a MAD
dataset (RMF20_4). These positions were further refined using mlphare [CCP4 suite
(7)]. The binding sites for iridium hexamine were not identical between the four copies
in the asymmetric unit, but all sites (10 total) were found within the RNA major groove
and occurred primarily at the junctions between double and triple helices. Different
methods of density modification [from CCP4 (7), Resolve (8), and CNS (9)] produced
slightly different maps, which were combined using SigmaA [CCP4 suite (7)]. SigmaA
maps allowed better location of heavy atoms from an additional MAD dataset
(RMF20_9). SigmaA combination of both datasets following individual mlphare
refinement produced an electron density map of sufficient quality to place a GAAA
tetraloop [modeled from the L1 ribozyme ligase structure 2OIU.pdb (10)] and ideal
helical stem using Coot (11). Iterative rounds of model-building and REFMAC
refinement [CCP4 suite (7)] led to placement of two non-NCS-related ENE structures.
The second NCS-related copy of the assembly was located by molecular replacement
using Phaser [CCP4 suite (7)]. Further rounds of model building [including building of
oligo(A) sequences], restrained refinement, manual modification, and TLS treatment (12)
led to the final structure. Additional electron density from water and/or ions was
observed. The simulated annealing composite omit map was calculated in CNS (9) using
only RNA coordinates and was used to further assign water/metal ions. The identity of
these molecules was not verified, and they are all listed as water in the final PDB file.

NMR spectroscopy

After gel purification (above), RNA samples were further purified over DEAE
Sepharose:DEAE Sephacel (1:1) to remove acrylamide and other impurities. Fractions
containing RNA were precipitated with ethanol, exchanged into a buffer of 5 mM
potassium cacodylate, pH 6.5, 50 mM KCl, 1 mM MgCl₂, 0.1 mM potassium EDTA, and
10% D2O, and then concentrated to 225–250 mL. Samples were heated to 95°C for 5
minutes, then snap-cooled on ice before transfer to Shigemi NMR tubes. For samples
containing the ENE:A10 complex, A10 RNA was added during the final buffer exchange
step. A 1.5 mM ENE core sample was used for the 2D NOESY spectrum (250 ms
mixing time), which was collected at 20°C. One-dimensional spectra of complex or ENE
alone were run at temperatures ranging from 10–40°C.

Gel Shift Assays

Trace 5´-[³²P] end-labeled A-rich oligonucleotides were mixed with binding
buffer (final concentration: 5 mM sodium cacodylate, 50 mM KCl, 1 or 10 mM MgCl₂,
and 0.5 mg/ml tRNA) or varying amounts of ENE RNA in binding buffer, heated at 95°C
for 3 minutes, and snap-cooled on ice. After equilibration at room temperature for at
least one hour, 2.5 mL 50% glycerol was added to each 10 mL reaction. Samples were
then separated on an 8% native tris-borate gel with 1 or 10 mM magnesium chloride in
the gel and running buffer. Electrophoresis was carried out for 2 hours at 200 V and 4°C.
Gels were dried and exposed to a phosphorimager screen.

Deadenylation assays

DNA templates were prepared by PCR and corresponded to the 327 3´-terminal
nucleotides of PAN RNA containing either wild-type or mutant ENE (DENE or U903C,
U949C) flanked by a T7 RNA polymerase binding site and poly(A) tail (A₆₀, A₅₇GA₂, or
A₁₉GA₄₀). Uniformly  [g³²P]-U-labeled RNAs were transcribed and purified by
denaturing gel electrophoresis. Deadenylation assays (30 mL) were conducted as in (13).
Reactions contained 40% Dignam nuclear extract (14), 0.5 mM MgCl₂, 36 ng/mL poly(A)
(Sigma), 3% polyvinyl alcohol, 8 mM HEPES, pH 7.9, 0.08 mM EDTA, 100 mM KCl,
0.8 units/mL RNase inhibitor (Roche), and approximately 2 nM RNA substrate.
Reactions were incubated at 30°C, and aliquots were removed at 15, 30, 45, and 60
minutes. Deadenylated RNA (A₀) was prepared by adding 1 mL of 40 mM oligo(dT)₄₀ to
reaction tubes after 60 minutes and allowing endogenous RNAse H present in the extract
to act for a further 30 minutes at 30°C. The 0 minute time point (A₆₀ size standard) was
not exposed to nuclear extract. Following Trizol extraction and RNA precipitation,
samples were separated on a 6% urea gel. Gels were dried and exposed to a
phosphorimager screen.

Structure figures were prepared and RMSDs were calculated in PyMOL (DeLano
Scientific).

A classical pseudoknot is a tertiary RNA interaction made by base pairing
between a hairpin loop and an external single-stranded region, forming two helical stems
S1 and S2 joined by three loops, L1–L3 (15, 16). Marked distributions have been noticed
for loop length and sequence distribution [reviewed in (17)]. L1 typically is short (~ 90%
are < 4 nt) and U-rich. It lies in the major groove of S2 and can be unstructured. In the
overwhelming majority of cases, L2 is 0 or 1 nt long, allowing coaxial stacking of S1 and
S2. L3 is more variable in length, but the majority of characterized pseudoknots have an
L3 that is < 6 nt, A-rich at the 3'´ end, and lying in the minor groove of S1. Asymmetry in
the length and base composition of loops L1 and L3 is attributed to the asymmetry
between the major and minor groove, which these loops cross, respectively.

Formally, the ENE interaction with the poly(A) tail of PAN RNA could be
described as a long-range pseudoknot with L1 = 47 nt, L2 = 2 nt, and an L3 of ~120 to
320 nt (depending on where the ENE bound along the poly(A) tail). Here, the loop L1
contains the upper stem of the ENE, while loop L3 contains the intervening sequence
between the ENE and the PAN RNA poly(A) tail. Despite the presence of a 2-nt L2, the
A and Hoogsten U strands of S2 stack upon S1.

Although the pseudoknots discussed in the text [from telomerase RNA (18) and
the SAM-II riboswitch (19)] contain major groove triple helices between L1 and S2,
many other structural variants exist. For instance, the classic example is the ribosomal
frameshifting pseudoknot from beet western yellow virus. In this 26 nt pseudoknot, L1 is
only 2 nt long, with the first nucleotide involved in a base quadruple interaction in the
major groove of S2, while L2 is 6 nt long and is involved in an extensive hydrogenbonded
minor groove triplex (20). A more recent example is the class I preQ1 riboswitch
(21, 22), which has a 2-nt L1 that makes Watson-Crick•Hoogsteen pairs with the last
nucleotides of L3, while other nucleotides in the 6-nt L3 make inclined adenosine
Hoogsteen interactions with S1. Metal ions are often observed in the vicinity of triplexes
in pseudoknots (23), but the thermodynamic significance of these ions is unclear (24).

Fig. S1. Interactions between oligo(A) and various ENE constructs were investigated using native gel shift assays with trace [³²P] 5´-end labeled A10 RNA.

(A) The ENE core contains the determinants for binding oligo(A) RNA. Gel shift assays were performed
either with truncated or full-length ENE and A₁₀ RNA. In the 39-nt truncated version used in this gel shift, a GCAA tetraloop with a G-C closing base pair replaced the upper hairpin of the ENE, and the sequence ended at U39. The 86-nt full-length ENE (FL ENE) used here contained the 79-nt ENE (shown in Fig. 1A) preceded by GGG to allow T7 polymerase transcription and followed by GGAU for plasmid linearization (the italicized nucleotides correspond to PAN RNA sequence). Similar results were obtained with all ENE constructs and AN oligonucleotides used for crystallization trials. 1 mM MgCl₂ was present in the binding buffer, gel, and running buffer.

(B) Secondary structure of the ENE core construct used for crystallography, highlighting the U to C mutations used for gel shift in (C).

(C) Single U to C mutations in the U-rich loop reduce the binding affinity between the ENE and A₁₀ more than 100-fold. Gel shift assays were performed using either the wild-type 40-nt ENE core shown in (B) or an RNA containing a single U to C substitution in the 5´ side of the U-rich loop (5´C) or in the 3´ side of the U-rich loop (3´C), as labeled in (B). To assess the reductions in binding due to these substitutions, the MgCl₂ concentration in the binding buffer, gel, and running buffer was increased to 10 mM. Under these conditions, >10-fold tighter binding was observed between the wild-type ENE core and A₁₀.

Fig. S2. The ENE core adopts a hairpin structure in solution.

Putative secondarystructure is shown on the left, with red and blue boxes highlighting
the upper and lower stems, respectively. The imino-imino region of a two-dimensional (2D)
1H-1H NOESY spectrum of the ENE core alone is shown on the right, aligned with the
imino region of a one-dimensional (1D) 1H spectrum above. Peak assignments were
made on the basis of the pattern of imino-imino NOEs, where red labels correspond
to the upper stem and blue to the lower stem.

(B) A10 binding adds complexity to the imino region of the 1D 1H
spectrum, presumably through addition of hydrogen bonds. Spectra were collected at
10°C. The irregular peak at about 11.2 ppm in the ENE alone spectrum disappears with
increasing temperatures and likely arises from U-U interactions in the unbound internal
loop. This peak did not give rise to any NOEs in the 2D NOESY spectrum above.

Fig. S3. (A) The full asymmetric unit of the crystal contains four ENE:A₉ complexes.

The complex described in the main text (Fig. 1, chains A and B in the PDB file) is shown
in the left foreground in green (ENE) and magenta (A₉), in front of its NCS-related
complex (PDB file chains E and F). The complexes on the right, shown in yellow (ENE)
and blue (A₉), are a second pair of NCS-related structures (chains C, D, G, and H), which
have a higher overall B_factor and a less well-defined A oligonucleotide. Crystal-packing
interactions occur between pairs of non-NCS-related complexes and involve the GAAA
tetraloops and the 5'´ ends of the A₉ oligonucleotide. Most notably, the A1 nucleotides of
the complex described in the text and its NCS-related complex are splayed out and stack
under the A1 nucleotides of the non-NCS-related complexes.

(B) Superposition of the two non-NCS-related complexes in the asymmetric unit [superposition done using all
non-hydrogen atoms in the triple helix, with colors as in (A)]. Crystal-packing interactions lead to differences in the orientations of the GAAA tetraloop and the 5´ end of the A₉ RNA. Crystal packing at the 5´-terminal A (above) appears to distort this strand in the second structure (in yellow and blue), such that only two A-minor
interactions are formed and a third A nucleotide is bulged out, with the base disordered.

Density can clearly be traced for only eight A nucleotides in this strand.

(B) The simulated annealing composite omit map calculated in CNS at 2.5 Å resolution (9), focusing on the U-A•U triple helix. The RNA models are shown (ENE in green and A₉ in magenta) superimposed on the electron density map contoured at 1.5 s (grey). The red map, contoured at 3.5 s, highlights the locations of the phosphate atoms.

(A) The complex between the ENE and A9 RNA (triple helix boxed).

(B) The crystal structure of the SAM-II riboswitch in complex with the ligand S-methionyladenosine (SAM) [PDB 2QWY, structure A (19)]. The SAM moiety is shown in stick representation. The triple helix (boxed) comprises two U-A•U base triples and three non-canonical base triples (A-U•U, where the A is from SAM; G-C•A; and CG•G). The strand contributing the central GCUAA nucleotides is shown in cyan, while the strands that base pair to this central strand are shown in brown.

(C) Superposition of the triple helices from (A) and (B), with colors and orientations as in (A) and (B).
Superposition of the top two U-A•U base triples of both structures gives an RMSD = 0.9 Å over all non-hydrogen atoms. Differences in sequence and backbone continuity between the two structures leads to a higher deviation over the entire triple helix (RMSD = 2.1 Å over all equivalent non-hydrogen atoms [excluding the SAM moiety and its equivalent nucleotide in the ENE:A₉ structure (U29), as well as non-identical bases in the lower three base triples]).

(A) Close-up of the major groove triple helix formed between the internal loop of the ENE (green) and five nucleotides of the A₉ RNA (magenta), with the bases involved in Watson-Crick pairing in yellow. U10 and U29 (U903 and U949, respectively, using PAN RNA numbering, circled in brown) contact the same A (A7) in
the crystal structure.

(B) As (A), but depicting the locations of U9C and U29C mutations (U902C and U949C, respectively, using PAN RNA numbering, circled in blue), which contact two different A nucleotides in the triple helix (A6 and A7, respectively).

(C) Native gel shift assay of in vitro-transcribed wild-type core ENE (left), U10C, U29C ENE (middle), or U9C, U29C ENE (right) with A₁₀ (top panel) or A₇GA₂ (bottom panel). 10 mM MgCl₂ was present in the binding buffer, gel, and running buffer. Although both double-mutant ENEs exhibit greatly reduced binding to A₁₀, the U10C, U29C ENE recovers full affinity with A₇GA₂, indicating formation of a C-G•C base triple. The U9C, U29C ENE, which is incapable of forming the C-G•C base triple, is not competent for binding A₇GA₂. Weak binding of the wild-type ENE core to A₇GA₂ is due either to formation of a weak U-G•U base triple or to a register shift in which the type III A-minor interaction would not be formed.

In this highly detailed study, Rachel Mitton-Fry, Suzanne DeGregorio, Jimin Wang, Thomas Steitz, and Joan Steitz have found that a viral noncoding RNA molecule from Kaposi's Sarcoma patients interacts with the duplex viral RNA to stabilize the virus against lysis. This may prolong the activity of the virus within the patient, and extend the lethality of the disease. A triplex RNA structure may underlie other viral neoplasms.

1. Each cell retains all of its embryonic genes for a lifetime.

2. Controls for embryonic genes are often absent in adults.

3. Uncontrolled embryonic genes can replicate wildly.

4.  Replicating genes participate in  intra-cellular competition.

5.  The basis for gene competition is selective transcription.

6.  MicroRNAs can reprogram embryomic transcription.

7.  Gene reprogramming can produce normal phenotypes.

8.  Normal phenotypes can by-pass chromosomal lesions.

9.  MicroRNA therapy may need to be permanent.

10. Transplantation of microRNAs could be preferred.

http://www.embryomas.net/

1. Pathways within cell genomes involve a flow of information.

2. Information can flow by direct contact or by third parties.

3. Direct contact within whole genomes is difficult to regulate.

4. DNA-DNA direct contects are influenced by agents.

5. Nuclear agents include hydrophilic ionic and hydrophobic conforming ligands.

6. Third parties within genomes involve RNAs and proteins.

7.  RNAs and proteins are easy to regulate or reverse.

8.  Information can be shared, lost, or transformed.

9. System information can be hidden during system isolation.

10.  Local information can be permanently lost during system entropy.

http://www.cancerbiophysics.net/

Links to Current Research in Euchromatin:
Links to Euchromatin Activator RNA Reviews:
Links to Euchromatin Activator RNA Research:
Links to Ultrastructural Probes of DNase I-Sensitive Sites:
Links to RNA as a Therapeutic Agent:
Links to Hodgkin Lymphoma Immuno-Pathology:
Links to Activated T-Lymphocyte Immunotherapy:
Links to Medical Systems Biology:
Links to Selective Gene Transcription:
Links to RNA-Induced Epigenetics:
Links to RNA-Induced Embryogenesis:
Links to RNA and Biological Causality:
Links to Reprogramming and Neoplasia:

A Brief History of Activator RNA:

"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA".
(PowerPoint Presentation).

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