Sm Site Research Articles

Effects of wind erosion on the spatial heterogeneity of soil nutrients in two desert grassland communities

Wind is known to affect the spatial heterogeneity of soil resources in arid and semiarid systems, but multi-year, quantified observations are largely absent. We studied the effects of wind erosion on the spatial distribution of soil organic carbon (SOC) and other soil nutrients at the Jornada Experimental Range, in southern New Mexico. Enhanced wind erosion was encouraged by grass cover reduction in a Sporobolus-mesquite dominated site (SM) and a Bouteloua-mesquite dominated site (BM). The scale and magnitude of spatial dependence for the soil analytes were quantified using geostatistical analyses. Results of this study show that soil organic matter related analytes such as SOC, TN, Navail, and SO4 2­ are among the first to be eroded and redistributed; cations such as Ca2+ and Mg2+ may not be removed and redistributed significantly; and other ions such as K+, Na+ and Cl− showed no discernible pattern of change. Geostatistics show that wind appeared to increase the scale of spatial autocorrelation, but decrease the scale of spatial dependence of most soil analytes over 2–3 windy seasons. In the wind enhanced plot of the SM site, up to 99% of the spatial dependence of SOC was autocorrelated at the distance of 1.45 m before the initiation of wind erosion, but the spatial dependence dropped significantly to only 60% at a larger autocorrelation distance of 2.76 m after three windy seasons. Similar but less significant changes were observed for SOC in the BM site. Despite the differential effects of wind on the soil analytes, we conclude that the overall results of wind on the grass cover reduction plots are the disappearance of small, strong fertile islands, which may be related to grasses; and the reinforcement of large fertile islands, which are likely related to mesquite shrubs. In addition, the change of the spatial patterns of SOC and other soil nutrients induced by enhanced wind erosion may persist and reinforce soil islands associated with shrubs, thus allowing a positive feedback for further desertification in this arid grassland.

Rare-earth site splitting in Sm3MoO7

Trisamarium molybdenum heptaoxide, Sm(3)MoO(7), is isomorphous with Ln(3)MoO(7) (Ln = La and Pr). The crystal structure consists of chains of corner-linked MoO(6) octahedra running parallel to the b axis and separated from each other by seven- or eight-coordinate Sm-O polyhedra. In contrast to La(3)MoO(7) and Pr(3)MoO(7), a splitting of one Sm site into two positions is observed.

The Gemin5 Protein of the SMN Complex Identifies snRNAs

The survival of motor neurons protein (SMN) is part of a large complex that contains six other proteins, Gemins2-7. The SMN complex assembles the heptameric Sm protein core on small nuclear RNAs (snRNAs) and plays a critical role in the biogenesis of snRNPs, the major and essential components of mRNA splicing in eukaryotes. For its function, the SMN complex binds Sm proteins and snRNAs, which it distinguishes from other RNAs by specific features they contain. We show here that Gemin5, a 170 kDa WD-repeat protein, is the snRNA binding protein of the SMN complex. Gemin5 binds directly and specifically to the unique features, including the Sm site, of snRNAs. Reduction of Gemin5 results in reduced capacity of the SMN complex to bind snRNAs and to assemble Sm cores. Gemin5 therefore functions as the factor that allows the SMN complex to distinguish snRNAs from other cellular RNAs for snRNP biogenesis.

In vivo assembly of functional U7 snRNP requires RNA backbone flexibility within the Sm-binding site

Most histone precursor mRNAs (pre-mRNAs) in metazoans are matured by 3'-end cleavage directed by the U7 small nuclear ribonucleoprotein (snRNP). RNA functional groups necessary for in vivo assembly and activity of the U7 snRNP were examined by nucleotide-analog interference mapping and mutagenesis using a chimeric mouse histone H4 pre-mRNA-U7 snRNA construct that is cleaved in cis in Xenopus laevis oocytes. Assembly of the unique U7 Sm protein core is rate limiting for processing in vivo and requires four conserved nucleotides within the U7 Sm-binding site, as well as the correct positioning and size of the U7 terminal stem-loop structure. To our surprise, pseudouridine substitution revealed a requirement for backbone flexibility at a particular position within the U7 Sm site, providing in vivo biochemical evidence that an unusual C2'-endo sugar conformation is necessary for assembly of the Sm ring.

RNA determinants and protein components of the histone pre‐mRNA processing machinery

Most metazoan mRNAs encoding histones are cleaved but not polyadenylated at their 3′ ends. Cleavage is directed by the U7 small nuclear ribonucleoprotein (U7 snRNP) and requires a heat-labile factor. We recently identified the heat-sensitive complex essential for histone pre-mRNA cleavage. It includes all five subunits of the cleavage and polyadenylation specificity factor, two subunits of the cleavage stimulation factor and symplekin. To identify which of these factors are essential for cleavage, we are attempting to reconstitute an in vitro processing system with purified and recombinant components. To investigate the role of U7 snRNA in processing, we have determined the RNA requirements for the assembly of a functional U7 snRNP in the Xenopus oocyte system by injecting chimeric histone H4 pre-mRNA / U7 snRNA constructs. We find that the rate limiting step is the assembly of the unique U7 Sm core of proteins, consistent with a role of U7 snRNP in recruiting the additional components of the processing machinery. By Nucleotide Analog Interference Mapping (NAIM) and conventional mutagenesis, we have identified the sequence and structural features of U7 critical for assembly of active particles. These include a flexible 5′-half (revealed by pseudouridine substitution) and four critical nucleotides in the 3′-half of the U7 Sm site, as well as correct placement and length of the U7 hairpin. Research support: HHMI, NIH.

The SMN Complex: An Assembly Machine for RNPs

In eukaryotic cells, the biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and likely other RNPs is mediated by an assemblyosome, the survival of motor neurons (SMN) complex. The SMN complex, composed of SMN and the Gemins (2-7), binds to the Sm proteins and to snRNAs and constructs the heptameric rings, the common cores of Sm proteins, on the Sm site (AU(56)G) of the snRNAs. We have determined the specific sequence and structural features of snRNAs for binding to the SMN complex and Sm core assembly. The minimal SMN complex-binding domain in snRNAs (except U1) is composed of an Sm site and a closely adjacent 3'stem-loop. Remarkably, the specific sequence of the stemloop is not important for SMN complex binding, but it must be located within a short distance of the 3'end of the RNA for an Sm core to assemble. This minimal snRNA-defining "snRNP code" is recognized by the SMN complex, which binds to it directly and with high affinity and assembles the Sm core. The recognition of the snRNAs is provided by Gemin5, a component of the SMN complex that directly binds the snRNP code. Gemin5 is a novel RNA-binding protein that is critical for snRNP biogenesis. Thus, the SMN complex is the identifier, as well as assembler, of the abundant class of snRNAs in cells. The function of the SMN complex, previously unanticipated because RNP biogenesis was believed to occur by self-assembly, confers stringent specificity on otherwise potentially illicit RNA-protein interactions.

Specific Sequence Features, Recognized by the SMN Complex, Identify snRNAs and Determine Their Fate as snRNPs

The survival of motor neurons (SMN) complex is essential for the biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) as it binds to and delivers Sm proteins for assembly of Sm cores on the abundant small nuclear RNAs (snRNAs). Using the conserved snRNAs encoded by the lymphotropic Herpesvirus saimiri (HVS), we determined the specific sequence and structural features of RNAs for binding to the SMN complex and for Sm core assembly. We show that the minimal SMN complex-binding domain in snRNAs, except U1, is comprised of an Sm site (AUUUUUG) and an adjacent 3' stem-loop. The adenosine and the first and third uridines of the Sm site are particularly critical for binding of the SMN complex, which directly contacts the backbone phosphates of these uridines. The specific sequence of the adjacent stem (7 to 12 base pairs)-loop (4 to 17 nucleotides) is not important for SMN complex binding, but it must be located within a short distance of the 3' end of the RNA for an Sm core to assemble. Importantly, these defining characteristics are discerned by the SMN complex and not by the Sm proteins, which can bind to and assemble on an Sm site sequence alone. These findings demonstrate that the SMN complex is the identifier, as well as assembler, of the abundant class of snRNAs in cells because it is able to recognize an snRNP code that they contain.

5-hydroxytryptamine 1A (5-HT 1A) but not 5-HT 3 receptor is involved in mediating the nucleus submedius 5-HT-evoked antinociception in the rat

Our previous studies have indicated that the thalamic nucleus submedius (Sm) is involved in modulation of nociception as part of an ascending component of an endogenous analgesic system consisting of spinal cord-Sm-ventrolateral orbital cortex (VLO)-periaqueductal gray (PAG)-spinal cord loop. Microinjection of 5-hydroxytryptamine (5-HT) into Sm produces antinociception and this effect is blocked by 5-HT 2 receptor antagonist. The aim of the present study was to examine whether the 5-HT 1 and 5-HT 3 receptors were also involved in the Sm 5-HT-evoked antinociception. Nociception was assessed in lightly anesthetized rats with radiant-heat-evoked tail flick (TF). 5-HT 1A and 5-HT 3 receptor antagonists were microinjected into the Sm alone or in combination with a microinjection of 5-HT into the same Sm site. 5-HT 1A receptor antagonist p-MPPI (0.87 nmol) facilitated the TF reflex; a lower dose (0.43 nmol) of p-MPPI significantly attenuated the Sm 5-HT-evoked inhibition of TF reflex. Microinjection of the 5-HT 3 receptor antagonist LY-278,584 (12 nmol) had no effect either on the TF reflex or on the Sm 5-HT-evoked inhibition. These results suggest that 5-HT 1A receptor but not 5-HT 3 receptor is involved in mediating the 5-HT-evoked antinociception. Possible mechanisms of Sm 5-HT-induced descending antinociception are discussed.

Temperature and field dependence of the spin magnetization density inSmMn2Ge2

The site specific spin moments in the layered intermetallic compound $\mathrm{Sm}{\mathrm{Mn}}_{2}{\mathrm{Ge}}_{2}$ have been measured directly as a function of field, between 0.02 and 1 T, using the spin-polarized Compton scattering technique at 13, 40, and 230 K. The field dependence of the orbital moment has been determined by comparison at each temperature with magnetometry data. A small finite Sm spin moment is observed at a low applied field of 0.02 T. These data indicate that in the high-temperature ordered phase the rare-earth sites exhibit a small net moment and exhibit paramagnetic behavior with little coupling to the Mn spin sublattice. Our low-temperature, low-field data indicate a change in magnetic order of the Sm site at $\ensuremath{\approx}35\phantom{\rule{0.3em}{0ex}}\mathrm{K}$ that is metamagnetic in nature. However, we find no evidence for a zero-field transition in the Sm sublattice as previously suggested.

SnRNAs contain specific SMN-binding domains that are essential for snRNP assembly.

To serve in its function as an assembly machine for spliceosomal small nuclear ribonucleoprotein particles (snRNPs), the survival of motor neurons (SMN) protein complex binds directly to the Sm proteins and the U snRNAs. A specific domain unique to U1 snRNA, stem-loop 1 (SL1), is required for SMN complex binding and U1 snRNP Sm core assembly. Here, we show that each of the major spliceosomal U snRNAs (U2, U4, and U5), as well as the minor splicing pathway U11 snRNA, contains a domain to which the SMN complex binds directly and with remarkable affinity (low nanomolar concentration). The SMN-binding domains of the U snRNAs do not have any significant nucleotide sequence similarity yet they compete for binding to the SMN complex in a manner that suggests the presence of at least two binding sites. Furthermore, the SMN complex-binding domain and the Sm site are both necessary and sufficient for Sm core assembly and their relative positions are critical for snRNP assembly. These findings indicate that the SMN complex stringently scrutinizes RNAs for specific structural features that are not obvious from the sequence of the RNAs but are required for their identification as bona fide snRNAs. It is likely that this surveillance capacity of the SMN complex ensures assembly of Sm cores on the correct RNAs only and prevents illicit, potentially deleterious, assembly of Sm cores on random RNAs.

Involvement of GABAergic modulation of the nucleus submedius (Sm) morphine-induced antinociception

Previous studies have shown that microinjection of morphine into the nucleus submedius (Sm) of the thalamus produces antinociception. The aim of the current study was to examine whether γ-aminobutyric acid (GABA)ergic terminals in the Sm were involved in this antinociception. Under light anesthesia, the GABA A receptor antagonist bicuculline or agonist muscimol was microinjected into the Sm of the thalamus in Sm non-morphine-treated (control) or Sm morphine-treated (microinjection into the Sm in the thalamus) rats. Tail flick latencies (TFL) were measured in each of these groups of rats every 5 min. Bicuculline (100, 200, 500 ng in 0.5 μl) depressed the TF reflex in a dose-dependent fashion, and this effect was blocked by microinjection of the opioid receptor antagonist naloxone (0.5 μg) into the same Sm site. A small dose (100 ng) of bicuculline microinjected into Sm significantly enhanced the morphine-evoked inhibition of TF reflex. In contrast, administration of muscimol (250 ng) did not significantly influence the TF reflex in Sm non-morphine-treated rats, but it significantly attenuated the morphine-induced antinociception in the Sm morphine-treated rats. These results suggest that locally released GABA acting at GABA A receptors is involved in the modulation of Sm morphine-induced antinociception, and support the hypothesis that a disinhibitory effect elicited by morphine on GABAergic terminals in Sm may lead to activation of the Sm-ventrolateral orbital cortex (VLO)–perioqueductal gray (PAG) brainstem descending inhibitory system and depression of the nociceptive inputs at the spinal cord level.

Effect of lattice relaxation on magnetic anisotropy:Zr-dopedSm2Co17

The magnetic anisotropy energy (MAE) in rare-earth/transition-metal hard magnets originates from the onsite anisotropy of the rare-earth element with additional contribution from the transition-metal sublattice. While ${\mathrm{SmCo}}_{5}$ can be transformed to ${\mathrm{Sm}}_{2}{\mathrm{Co}}_{17}$ by partial substitution of Sm by ${\mathrm{Co}}_{2}$ dumbbells, the MAE in ${\mathrm{Sm}}_{2}{\mathrm{Co}}_{17}$ is noticeably smaller compared to that in ${\mathrm{SmCo}}_{5}.$ However, small dopings of nonmagnetic Zr significantly increase the MAE in ${\mathrm{Sm}}_{2}{\mathrm{Co}}_{17}.$ We show that the changes to the MAE in ${\mathrm{Sm}}_{2}{\mathrm{Co}}_{17}$ can be traced down to the reduction of the crystal field at the Sm site due to the Co sublattice relaxation upon $\mathrm{Sm}\ensuremath{\rightarrow}{\mathrm{Co}}_{2}$ substitution which is subsequently restored upon substitution of ${\mathrm{Co}}_{2}\ensuremath{\rightarrow}\mathrm{Zr}.$

The roles of different types of glutamate receptors involved in the mediation of nucleus submedius (Sm) glutamate-evoked antinociception in the rat

Based on our previous findings that glutamate microinjected into the thalamic nucleus submedius (Sm) inhibits dose-dependently the rat tail-flick (TF) reflex, this study investigated which glutamate receptor subtype is involved in mediating this effect. The effects of an NMDA ( N-methyl- d-aspartate), non-NMDA or metabotropic glutamate receptor (mGluR) antagonist microinjected into Sm on the TF reflex were examined in untreated or in Sm glutamate treated (microinjection into the Sm) rats. The TF latencies were measured in each of these groups of rats every 5 min. Injection of DNQX [6,7-dinitroquinoxaline-2,3(1H,4H)-dione], a non-NMDA receptor antagonist, or (±)-MCPG [(±)-α-methyl-4-carboxyphenylglycine], a mGluR antagonist, into the Sm blocked the inhibitory effects induced by a subsequent microinjection of glutamate into the same Sm site. The TF latency increased only by 6.6±1.6 or 9.0±1.1%, respectively, of the baseline value, which was markedly less than that (51.3±8.4 or 50.7±5.3%) obtained from injection of glutamate only ( P<0.001, n=8). However, pre-microinjection of MK-801 [(+)-5-methyl-10,11-dibenzo[ a, d]cyclohepten-5,10-imine], an NMDA receptor antagonist, into the Sm had no effect on the Sm glutamate-evoked inhibition of the TF reflex. The TF latency change (40.0±11.1%) was not significantly different ( P>0.05, n=8) compared with that obtained from glutamate injection alone. These observations suggest that non-NMDA and metabotropic glutamate receptors, but not NMDA receptors, are involved in mediating Sm glutamate-evoked antinociception.

On the Role of Exon and Intron Sequences intrans-Splicing Utilization and cap 4 Modification of the Trypanosomatid Leptomonas collosoma SL RNA

In trypanosomatid protozoa the biogenesis of mature mRNA involves addition of the spliced leader (SL) sequence from the SL RNA to polycistronic pre-mRNA via trans-splicing. Here we present a mutational analysis of the trypanosomatid Leptomonas collosoma SL RNA to further our understanding of its functional domains important for trans-splicing utilization. Mutant SL RNAs were analyzed for defects in modification of the hypermethylated cap structure (cap 4) characteristic of trypanosomatid SL RNAs, for defects in the first step of the reaction and overall utilization in trans-splicing. Single substitution of the cap 4 nucleotides led to undermethylation of the cap 4 structure, and these mutants were all impaired in their utilization in trans-splicing. Abrogation of the sequence of the Sm-like site and sequences downstream to it also showed cap modification and trans-splicing defects, thus providing further support for a functional linkage between cap modifications and trans-splicing. Further, we report that in L. collosoma both the exon and intron of the SL RNA contribute information for efficient function of the SL RNA in trans-splicing. This study, however, did not provide support for the putative SL RNA-U6 small nuclear RNA (snRNA) interaction at the Sm site like in the nematodes, suggesting differences in the bridging role of U6 in the two trans-splicing systems.

Morphine microinjections into the rat nucleus submedius depress nociceptive behavior in the formalin test

Our previous studies have indicated that the thalamic nucleus submedius (Sm) is involved in modulation of nociception and plays an important role in an endogenous analgesic system (a feedback loop) consisting of spinal cord–Sm–ventrolateral orbital cortex–periaqueductal gray–spinal cord. To investigate whether opioids are involved in this antinociception pathway, the effects of microinjection of morphine and naloxone into the Sm on the nociceptive behavior (agitation) evoked in the formalin test were investigated in the awake rat using an automated movement detection system. The results indicate that a unilateral microinjection of morphine (5 μg, 0.5 μl) into the Sm suppresses the formalin-induced agitation response, but does not influence spontaneous motor activity, and that the morphine-induced depression can be reversed by microinjection of the opioid receptor antagonist naloxone (1.0 μg, 0.5 μl) into the same Sm site. The results suggest that opioid receptors in the Sm may be involved in the Sm-mediated depression of persistent inflammatory pain.

EFFECT OF 5-FU SUBSTITUTION AND MUTATION ON Sm PROTEIN BINDING TO HUMAN U4 snRNA

ABSTRACT The effects of native and non-native nucleotide substitution on the binding of Sm proteins to human U4 snRNA were investigated to determine if the Sm site was a likely target for the RNA-mediated effects of the anticancer drug 5-FU, and other nucleoside analogues. The Sm binding site of human U4 snRNA was prepared by in vitro transcription, and Sm protein binding was assessed using gel mobility shift assays. The U4:Sm RNA:protein complex was identified by immunoprecipitation with the Sm-specific Y12 antibody. The effects of 5-FU substitution were assessed by including FUTP in the in vitro transcription reactions. The effects of native nucleotide substitution were assessed by mutagenesis. Deletion mutants were used to assess the relative importance of the two stem-loops that flank the Sm binding site for protein binding. Point mutation (U→G) to the 5′-Urd in the Sm site reduced Sm protein binding while similar point mutation to the 3′-Urd had a lesser effect. Mutation (U→G) of all Urd in the Sm site completely inhibited Sm protein binding. The central stem-loop contributed significantly to Sm protein complex formation but the 3′ stem-loop had little effect. Substitution of Urd by 5-fluorourdine (FUrd) did not inhibit Sm protein binding, but reduced the stability of the resulting complex. The results indicate that 5-FU, or other Uracil analogues, are unlikely to exert RNA-mediated effects through inhibition of Sm protein binding.

Hole-doping effect on the Verwey-type transition and magnetoresistivity of Ba(Sm,Ca)Fe 2O 5+ δ

Two series of samples of the oxygen-deficient double-perovskite phase, Ba(Sm,Ca)Fe 2O 5+ δ were synthesized employing a sample encapsulation technique that utilizes Fe metal as an oxygen getter. The hole-doping level on the FeO 2 plane was controlled by varying the amount of excess oxygen or the Ca concentration at the Sm site. Earlier it had been found that at room temperature five-coordinated Fe 2+ and Fe 3+ species form pairs by sharing a d electron, leading to the formation of a Fe 2.5+ fluctuating valence state [Phys. Rev. B 60 (1999) 15,251]. At T V, a Verwey-type transition occurred, signifying the charge separation of the Fe 2.5+ fluctuating valence state into high-spin Fe 2+ and Fe 3+. Moreover, related to the Verwey-type transition a negative magnetoresistance peak with a magnitude of a few percent was observed [Appl. Phys. Lett. 77 (2000) 1683]. The position of the peak was found to correspond to the jumps seen in the susceptibility and resistivity vs. temperature curves. For the present samples, it is found that the value of T V is severely decreased upon increasing the Ca concentration, as seen by susceptibility and magnetoresistivity measurements. Introduction of excess oxygen leads to a less severe decrease of the transition temperature. The observed behavior of T V was reproduced by a simple model based on combinatorial-entropy calculations.

RNA binding in an Sm core domain: X-ray structure and functional analysis of an archaeal Sm protein complex.

Eukaryotic Sm and Sm-like proteins associate with RNA to form the core domain of ribonucleoprotein particles involved in pre-mRNA splicing and other processes. Recently, putative Sm proteins of unknown function have been identified in Archaea. We show by immunoprecipitation experiments that the two Sm proteins present in Archaeoglobus fulgidus (AF-Sm1 and AF-Sm2) associate with RNase P RNA in vivo, suggesting a role in tRNA processing. The AF-Sm1 protein also interacts specifically with oligouridylate in vitro. We have solved the crystal structures of this protein and a complex with RNA. AF-Sm1 forms a seven-membered ring, with the RNA interacting inside the central cavity on one face of the doughnut-shaped complex. The bases are bound via stacking and specific hydrogen bonding contacts in pockets lined by residues highly conserved in archaeal and eukaryotic Sm proteins, while the phosphates remain solvent accessible. A comparison with the structures of human Sm protein dimers reveals closely related monomer folds and intersubunit contacts, indicating that the architecture of the Sm core domain and RNA binding have been conserved during evolution.

Structure of the Sm Binding Site From Human U4 snRNA Derived From a 3 ns PME Molecular Dynamics Simulation

A molecular dynamics simulation of the Sm binding site from human U4 snRNA was undertaken to determine the conformational flexibility of this region and to identify RNA conformations that were important for binding of the Sm proteins. The RNA was fully-solvated (>9,000 water molecules) and charge neutralized by inclusion of potassium ions. A three nanosecond MD simulation was conducted using AMBER with long-range electrostatic forces considered using the particle mesh Ewald summation method. The initial model of the Sm binding site region had the central and 3′ stem-loops that flanked the Sm site co-axial with one another, and with the single-stranded Sm binding site region ([I] conformation). During the course of the trajectory, the axes of the 3′ stem-loop, and later the central stem-loop, became roughly orthogonal from their original anti-parallel orientation. As these conformational changes occurred, the snRNA adopted first an [L] conformation, and finally a [U] conformation. The [U] conformation was more stable than either the [I] or [L] conformations, and persisted for the final 1 ns of the trajectory. Analysis of the structure resulting from the MD simulations revealed the bulged nucleotide, U114, and the mismatched A91-G110 base pair provided distinctive structural features that may enhance Sm protein binding. Based on the results of the MD simulation and the available experimental data, we proposed a mechanism for the binding of the Sm protein sub-complexes to the snRNA. In this model, the D1/D2 and E/F/G Sm protein sub-complexes first bind the snRNA in the [U] conformation, followed by conformational re-arrangement to the [I] conformation and binding of the D3/B Sm protein sub-complex.

The box C/D motif directs snoRNA 5'-cap hypermethylation.

The 5'-cap structure of most spliceosomal small nuclear RNAs (snRNAs) and certain small nucleolar RNAs (snoRNAs) undergoes hypermethylation from a 7-methylguanosine to a 2,2, 7-trimethylguanosine structure. 5'-Cap hypermethylation of snRNAs is dependent upon a conserved sequence element known as the Sm site common to most snRNAs. Here we have performed a mutational analysis of U3 and U14 to determine the cis-acting sequences required for 5'-cap hypermethylation of Box C/D snoRNAs. We have found that both the conserved sequence elements Box C (termed C' in U3) and Box D are necessary for cap hypermethylation. Furthermore, the terminal stem structure that is formed by sequences that flank Box C (C' in U3) and Box D is also required. However, mutation of other conserved sequences has no effect on hypermethylation of the cap. Finally, the analysis of fragments of U3 and U14 RNAs indicates that the Box C/D motif, including Box C (C' in U3), Box D and the terminal stem, is capable of directing cap hypermethylation. Thus, the Box C/D motif, which is important for snoRNA processing, stability, nuclear retention, protein binding, nucleolar localization and function, is also necessary and sufficient for cap hypermethylation of these RNAs.