Structural and mechanistic insights into the function of Leishmania ribosome lacking a single pseudouridine modification.

Leishmania is the causative agent of cutaneous and visceral diseases affecting millions of individuals worldwide. Pseudouridine (Ψ), the most abundant modification on rRNA, changes during the parasite life cycle. Alterations in the level of a specific Ψ in helix 69 (H69) affected ribosome function. To decipher the molecular mechanism of this phenotype, we determine the structure of ribosomes lacking the single Ψ and its parental strain at ∼2.4-3 Å resolution using cryo-EM. Our findings demonstrate the significance of a single Ψ on H69 to its structure and the importance for its interactions with helix 44 and specific tRNAs. Our study suggests that rRNA modification affects translation of mRNAs carrying codon bias due to selective accommodation of tRNAs by the ribosome. Based on the high-resolution structures, we propose a mechanism explaining how the ribosome selects specific tRNAs.

A single pseudouridine on rRNA regulates ribosome structure and function in the mammalian parasite Trypanosoma brucei.

Trypanosomes are protozoan parasites that cycle between insect and mammalian hosts and are the causative agent of sleeping sickness. Here, we describe the changes of pseudouridine (Ψ) modification on rRNA in the two life stages of the parasite using four different genome-wide approaches. CRISPR-Cas9 knock-outs of all four snoRNAs guiding Ψ on helix 69 (H69) of the large rRNA subunit were lethal. A single knock-out of a snoRNA guiding Ψ530 on H69 altered the composition of the 80S monosome. These changes specifically affected the translation of only a subset of proteins. This study correlates a single site Ψ modification with changes in ribosomal protein stoichiometry, supported by a high-resolution cryo-EM structure. We propose that alteration in rRNA modifications could generate ribosomes preferentially translating state-beneficial proteins.

TLR7/8 stress response drives histiocytosis in SLC29A3 disorders.

Loss-of-function mutations in the lysosomal nucleoside transporter SLC29A3 cause lysosomal nucleoside storage and histiocytosis: phagocyte accumulation in multiple organs. However, little is known about the mechanism by which lysosomal nucleoside storage drives histiocytosis. Herein, histiocytosis in Slc29a3-/- mice was shown to depend on Toll-like receptor 7 (TLR7), which senses a combination of nucleosides and oligoribonucleotides (ORNs). TLR7 increased phagocyte numbers by driving the proliferation of Ly6Chi immature monocytes and their maturation into Ly6Clow phagocytes in Slc29a3-/- mice. Downstream of TLR7, FcRγ and DAP10 were required for monocyte proliferation. Histiocytosis is accompanied by inflammation in SLC29A3 disorders. However, TLR7 in nucleoside-laden splenic monocytes failed to activate inflammatory responses. Enhanced production of proinflammatory cytokines was observed only after stimulation with ssRNAs, which would increase lysosomal ORNs. Patient-derived monocytes harboring the G208R SLC29A3 mutation showed enhanced survival and proliferation in a TLR8-antagonist-sensitive manner. These results demonstrated that TLR7/8 responses to lysosomal nucleoside stress drive SLC29A3 disorders.

Damage sensing mediated by serine proteases Hayan and Persephone for Toll pathway activation in apoptosis-deficient flies.

The mechanisms by which the innate immune system senses damage have been extensively explored in multicellular organisms. In Drosophila, various types of tissue damage, including epidermal injury, tumor formation, cell competition, and apoptosis deficiency, induce sterile activation of the Toll pathway, a process that requires the use of extracellular serine protease (SP) cascades. Upon infection, the SP Spätzle (Spz)-processing enzyme (SPE) cleaves and activates the Toll ligand Spz downstream of two paralogous SPs, Hayan and Persephone (Psh). However, upon tissue damage, it is not fully understood which SPs establish Spz activation cascades nor what damage-associated molecules can activate SPs. In this study, using newly generated uncleavable spz mutant flies, we revealed that Spz cleavage is required for the sterile activation of the Toll pathway, which is induced by apoptosis-deficient damage of wing epidermal cells in adult Drosophila. Proteomic analysis of hemolymph, followed by experiments with Drosophila Schneider 2 (S2) cells, revealed that among hemolymph SPs, both SPE and Melanization Protease 1 (MP1) have high capacities to cleave Spz. Additionally, in S2 cells, MP1 acts downstream of Hayan and Psh in a similar manner to SPE. Using genetic analysis, we found that the upstream SPs Hayan and Psh contributes to the sterile activation of the Toll pathway. While SPE/MP1 double mutants show more impairment of Toll activation upon infection than SPE single mutants, Toll activation is not eliminated in these apoptosis-deficient flies. This suggests that Hayan and Psh sense necrotic damage, inducing Spz cleavage by SPs other than SPE and MP1. Furthermore, hydrogen peroxide, a representative damage-associated molecule, activates the Psh-Spz cascade in S2 cells overexpressing Psh. Considering that reactive oxygen species (ROS) were detected in apoptosis-deficient wings, our findings highlight the importance of ROS as signaling molecules that induce the activation of SPs such as Psh in response to damage.

PDGF-B secreted from skeletal muscle enhances myoblast proliferation and myotube maturation via activation of the PDGFR signaling cascade.

Myokines, secreted factors from skeletal muscle, act locally on muscle cells or satellite cells, which is important in regulating muscle mass and function. Here, we found platelet-derived growth factor subunit B (PDGF-B) is constitutively secreted from muscle cells without muscle contraction. Furthermore, PDGF-B secretion increased with myoblast to myotube differentiation. To examine the role of PDGF-B as a paracrine or autocrine myokine, myoblasts or myotubes were treated with PDGF-B. As a result, myoblast proliferation was significantly enhanced via several signaling pathways. Intriguingly, myotubes treated with PDGF-B showed enhanced maturation as indicated by their increased myotube diameter, myosin heavy chain expression, and strengthened contractile force. These findings suggest that PDGF-B is constitutively secreted by myokines to enhance myoblast proliferation and myotube maturation, which may contribute to skeletal muscle regeneration.

DKC1 Overexpression Induces a More Aggressive Cellular Behavior and Increases Intrinsic Ribosomal Activity in Immortalized Mammary Gland Cells.

Dyskerin is a nucleolar protein involved in the small nucleolar RNA (snoRNA)-guided pseudouridylation of specific uridines on ribosomal RNA (rRNA), and in the stabilization of the telomerase RNA component (hTR). Loss of function mutations in DKC1 causes X-linked dyskeratosis congenita, which is characterized by a failure of proliferating tissues and increased susceptibility to cancer. However, several tumors show dyskerin overexpression. We observed that patients with primary breast cancers with high dyskerin levels are more frequently characterized by shorter survival rates and positive lymph node status than those with tumors with a lower dyskerin expression. To functionally characterize the effects of high dyskerin expression, we generated stably overexpressing DKC1 models finding that increased dyskerin levels conferred a more aggressive cellular phenotype in untransformed immortalized MCF10A cells. Contextually, DKC1 overexpression led to an upregulation of some snoRNAs, including SNORA67 and a significantly increased U1445 modification on 18S rRNA, the known target of SNORA67. Lastly, we found that dyskerin overexpression strongly enhanced the synthetic activity of ribosomes increasing translational efficiency in MCF10A. Altogether, our results indicate that dyskerin may sustain the neoplastic phenotype from an early stage in breast cancer endowing ribosomes with an augmented translation efficiency.

Cryo-EM structure of the highly atypical cytoplasmic ribosome of Euglena gracilis.

Ribosomal RNA is the central component of the ribosome, mediating its functional and architectural properties. Here, we report the cryo-EM structure of a highly divergent cytoplasmic ribosome from the single-celled eukaryotic alga Euglena gracilis. The Euglena large ribosomal subunit is distinct in that it contains 14 discrete rRNA fragments that are assembled non-covalently into the canonical ribosome structure. The rRNA is substantially enriched in post-transcriptional modifications that are spread far beyond the catalytic RNA core, contributing to the stabilization of this highly fragmented ribosome species. A unique cluster of five adenosine base methylations is found in an expansion segment adjacent to the protein exit tunnel, such that it is positioned for interaction with the nascent peptide. As well as featuring distinctive rRNA expansion segments, the Euglena ribosome contains four novel ribosomal proteins, localized to the ribosome surface, three of which do not have orthologs in other eukaryotes.

Direct Determination of Pseudouridine in RNA by Mass Spectrometry Coupled with Stable Isotope Labeling.

Pseudouridine (Ψ) is the only "mass-silent" nucleoside produced by post-transcriptional RNA modification. We developed a mass spectrometry (MS)-based technique coupled with in vivo deuterium (D) labeling of uridines for direct determination of Ψs in cellular RNA and applied it to the comprehensive analysis of post-transcriptional modifications in human ribosomal RNAs. The method utilizes human TK6/mouse FM3A cells deficient in uridine monophosphate synthase using a CRISPR-Cas9 technique to turn off de novo uridine synthesis and fully labels uridines with D at uracil positions 5 and 6 by cultivating the cells in a medium containing uridine-5,6-D2. The pseudouridylation reaction in those cells results in the exchange of the D at the C5 of uracil with hydrogen from solvent, which produces a -1 Da mass shift, thus allowing MS-based determination of RNA Ψs. We present here the experimental details of this method and show that it allows the identification of all Ψs in human major nuclear and nucleolar RNAs, including several previously unknown Ψs. Because the method allows direct determination of Ψs at the femtomole level of RNA, it will serve as a useful tool for structure/function studies of a wide variety of noncoding RNAs.

Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping.

N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA1-3. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac4C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac4C at single-nucleotide resolution. In human and yeast mRNAs, ac4C sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac4C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease4-6.

Method for Direct Mass-Spectrometry-Based Identification of Monomethylated RNA Nucleoside Positional Isomers and Its Application to the Analysis of Leishmania rRNA.

RNA post-transcriptional modifications are common in all kingdoms of life and are predominantly affiliated with methylations at various nucleobase positions. Methylations occur frequently at specific sites on the RNA nucleobases and appear to regulate site-specific intermolecular/intramolecular interactions. Herein, we present a method that utilizes liquid chromatography-mass spectrometry (LC-MS) to identify positional monomethylated RNA nucleoside isomers. The method produces profiles of in-source fragmentation and subsequent tandem mass spectrometry (MS2) (pseudo-MS3) of RNase-digested fragments of an RNA and distinguishes between positional methylated nucleobase isomers by comparing their intranucleobase fragment ion profiles with signature profiles derived from authentic isomers. For method validation, we independently determined the positions of all known monomethylated nucleoside isomers in the Escherichia coli 16S/23S rRNAs. As proof of concept, we further applied this technology to fully characterize the base-modified nucleoside positional isomers, in rRNAs derived from Leishmania donovani, a human blood parasite afflicting millions around the globe. The method described herein will be highly beneficial for the delineation of RNA modification profiles in various cellular RNAs, and as it only requires a subpicomole amount of RNA, it could also be used for the structure-function studies of RNA populations represented in minute amounts in the cell.

LYAR potentiates rRNA synthesis by recruiting BRD2/4 and the MYST-type acetyltransferase KAT7 to rDNA.

Activation of ribosomal RNA (rRNA) synthesis is pivotal during cell growth and proliferation, but its aberrant upregulation may promote tumorigenesis. Here, we demonstrate that the candidate oncoprotein, LYAR, enhances ribosomal DNA (rDNA) transcription. Our data reveal that LYAR binds the histone-associated protein BRD2 without involvement of acetyl-lysine-binding bromodomains and recruits BRD2 to the rDNA promoter and transcribed regions via association with upstream binding factor. We show that BRD2 is required for the recruitment of the MYST-type acetyltransferase KAT7 to rDNA loci, resulting in enhanced local acetylation of histone H4. In addition, LYAR binds a complex of BRD4 and KAT7, which is then recruited to rDNA independently of the BRD2-KAT7 complex to accelerate the local acetylation of both H4 and H3. BRD2 also helps recruit BRD4 to rDNA. By contrast, LYAR has no effect on rDNA methylation or the binding of RNA polymerase I subunits to rDNA. These data suggest that LYAR promotes the association of the BRD2-KAT7 and BRD4-KAT7 complexes with transcription-competent rDNA loci but not to transcriptionally silent rDNA loci, thereby increasing rRNA synthesis by altering the local acetylation status of histone H3 and H4.

Low-fluence blue light-induced phosphorylation of Zmphot1 mediates the first positive phototropism.

Phototropin1 (phot1) perceives low- to high-fluence blue light stimuli and mediates both the first and second positive phototropisms. High-fluence blue light is known to induce autophosphorylation of phot1, leading to the second positive phototropism. However, the phosphorylation status of phot1 by low-fluence blue light that induces the first positive phototropism had not been observed. Here, we conducted a phosphoproteomic analysis of maize coleoptiles to investigate the fluence-dependent phosphorylation status of Zmphot1. High-fluence blue light induced phosphorylation of Zmphot1 at several sites. Notably, low-fluence blue light significantly increased the phosphorylation level of Ser291 in Zmphot1. Furthermore, Ser291-phosphorylated and Ser369Ser376-diphosphorylated peptides were found to be more abundant in the low-fluence blue light-irradiated sides than in the shaded sides of coleoptiles. The roles of these phosphorylation events in phototropism were explored by heterologous expression of ZmPHOT1 in the Arabidopsis thaliana phot1phot2 mutant. The first positive phototropism was restored in wild-type ZmPHOT1-expressing plants; however, plants expressing S291A-ZmPHOT1 or S369AS376A-ZmPHOT1 showed significantly reduced complementation rates. All transgenic plants tested in this study exhibited a normal second positive phototropism. These findings provide the first indication that low-fluence blue light induces phosphorylation of Zmphot1 and that this induced phosphorylation is crucial for the first positive phototropism.

TDP-43 regulates site-specific 2'-O-methylation of U1 and U2 snRNAs via controlling the Cajal body localization of a subset of C/D scaRNAs.

TDP-43 regulates cellular levels of Cajal bodies (CBs) that provide platforms for the assembly and RNA modifications of small nuclear ribonucleoproteins (snRNPs) involved in pre-mRNA splicing. Alterations in these snRNPs may be linked to pathogenesis of amyotrophic lateral sclerosis. However, specific roles for TDP-43 in CBs remain unknown. Here, we demonstrate that TDP-43 regulates the CB localization of four UG-rich motif-bearing C/D-box-containing small Cajal body-specific RNAs (C/D scaRNAs; i.e. scaRNA2, 7, 9 and 28) through the direct binding to these scaRNAs. TDP-43 enhances binding of a CB-localizing protein, WD40-repeat protein 79 (WDR79), to a subpopulation of scaRNA2 and scaRNA28; the remaining population of the four C/D scaRNAs was localized to CB-like structures even with WDR79 depletion. Depletion of TDP-43, in contrast, shifted the localization of these C/D scaRNAs, mainly into the nucleolus, as well as destabilizing scaRNA2, and reduced the site-specific 2'-O-methylation of U1 and U2 snRNAs, including at 70A in U1 snRNA and, 19G, 25G, 47U and 61C in U2 snRNA. Collectively, we suggest that TDP-43 and WDR79 have separate roles in determining CB localization of subsets of C/D and H/ACA scaRNAs.

FANCD2 protects genome stability by recruiting RNA processing enzymes to resolve R-loops during mild replication stress.

R-loops, which consist of DNA : RNA hybrids and displaced single-strand DNA, are a major threat to genome stability. We have previously reported that a key Fanconi anemia protein, FANCD2, accumulates on large fragile genes during mild replication stress in a manner depending on R-loops. In this study, we found that FANCD2 suppresses R-loop levels. Furthermore, we identified FANCD2 interactions with RNA processing factors, including hnRNP U and DDX47. Our data suggest that FANCD2, which accumulates with R-loops in chromatin, recruits these factors and thereby promotes efficient processing of long RNA transcripts. This may lead to a reduction in transcription-replication collisions, as detected by PLA between PCNA and RNA Polymerase II, and hence, lowered R-loop levels. We propose that this mechanism might contribute to maintenance of genome stability during mild replication stress.

Subunit Composition of Ribosome in the yqgF Mutant Is Deficient in pre-16S rRNA Processing of Escherichia coli.

Escherichia coli 16S, 23S, and 5S ribosomal RNAs (rRNAs) are transcribed as a single primary transcript, which is subsequently processed into mature rRNAs by several RNases. Three RNases (RNase III, RNase E, and RNase G) were reported to function in processing the 5'-leader of precursor 16S rRNA (pre-16S rRNA). Previously, we showed that a novel essential YqgF is involved in that processing. Here we investigated the ribosome subunits of the yqgFts mutant by LC-MS/MS. The mutant ribosome had decreased copy numbers of ribosome protein S1, suggesting that the yqgF gene enables incorporation of ribosomal protein S1 into ribosome by processing of the 5'-end of pre-16S rRNA. The ribosome protein S1 is essential for translation in E. coli; therefore, our results suggest that YqgF converts the inactive form of newly synthesized ribosome into the active form at the final step of ribosome assembly.

Structural Analyses of Toll-like Receptor 7 Reveal Detailed RNA Sequence Specificity and Recognition Mechanism of Agonistic Ligands.

Toll-like receptor 7 (TLR7) is an innate immune receptor for single-stranded RNA (ssRNA) and has important roles in infectious diseases. We previously reported that TLR7 shows synergistic activation in response to two ligands, guanosine and ssRNA. However, the specific ssRNA sequence preference, detailed recognition mode of TLR7 and its ligand, and molecular determinants of TLR7 and TLR8 selectivity remain unknown. Here, we report on TLR7 from a large-scale crystallographic study combined with a multifaceted approach. We reveal that successive uridine-containing ssRNAs fully or moderately bind TLR7, whereas single uridine-containing ssRNAs have reduced affinities. We also reveal the detailed relationships between the chemical structures of ligands and their binding to TLR7. We demonstrate that an engineered TLR8 mutant alters its responsiveness to TLR7-specific ligands. Finally, we identify guanosine 2',3'-cyclic phosphate (2',3'-cGMP) as a possible endogenous ligand for TLR7 with greater affinity than guanosine. The abundant structural information will facilitate future development of treatments targeting TLR7.

Landscape of the complete RNA chemical modifications in the human 80S ribosome.

During ribosome biogenesis, ribosomal RNAs acquire various chemical modifications that ensure the fidelity of translation, and dysregulation of the modification processes can cause proteome changes as observed in cancer and inherited human disorders. Here, we report the complete chemical modifications of all RNAs of the human 80S ribosome as determined with quantitative mass spectrometry. We assigned 228 sites with 14 different post-transcriptional modifications, most of which are located in functional regions of the ribosome. All modifications detected are typical of eukaryotic ribosomal RNAs, and no human-specific modifications were observed, in contrast to a recently reported cryo-electron microscopy analysis. While human ribosomal RNAs appeared to have little polymorphism regarding the post-transcriptional modifications, we found that pseudouridylation at two specific sites in 28S ribosomal RNA are significantly reduced in ribosomes of patients with familial dyskeratosis congenita, a genetic disease caused by a point mutation in the pseudouridine synthase gene DKC1. The landscape of the entire epitranscriptomic ribosomal RNA modifications provides a firm basis for understanding ribosome function and dysfunction associated with human disease.

Replication stress induces accumulation of FANCD2 at central region of large fragile genes.

During mild replication stress provoked by low dose aphidicolin (APH) treatment, the key Fanconi anemia protein FANCD2 accumulates on common fragile sites, observed as sister foci, and protects genome stability. To gain further insights into FANCD2 function and its regulatory mechanisms, we examined the genome-wide chromatin localization of FANCD2 in this setting by ChIP-seq analysis. We found that FANCD2 mostly accumulates in the central regions of a set of large transcribed genes that were extensively overlapped with known CFS. Consistent with previous studies, we found that this FANCD2 retention is R-loop-dependent. However, FANCD2 monoubiquitination and RPA foci formation were still induced in cells depleted of R-loops. Interestingly, we detected increased Proximal Ligation Assay dots between FANCD2 and R-loops following APH treatment, which was suppressed by transcriptional inhibition. Collectively, our data suggested that R-loops are required to retain FANCD2 in chromatin at the middle intronic region of large genes, while the replication stress-induced upstream events leading to the FA pathway activation are not triggered by R-loops.

S1P-S1PR2 Axis Mediates Homing of Muse Cells Into Damaged Heart for Long-Lasting Tissue Repair and Functional Recovery After Acute Myocardial Infarction.

RATIONALE: Multilineage-differentiating stress enduring (Muse) cells, pluripotent marker stage-specific embryonic antigen-3+ cells, are nontumorigenic endogenous pluripotent-like stem cells obtainable from various tissues including the bone marrow. Their therapeutic efficiency has not been validated in acute myocardial infarction. OBJECTIVE: The main objective of this study is to clarify the efficiency of intravenously infused rabbit autograft, allograft, and xenograft (human) bone marrow-Muse cells in a rabbit acute myocardial infarction model and their mechanisms of tissue repair. METHODS AND RESULTS: In vivo dynamics of Nano-lantern-labeled Muse cells showed preferential homing of the cells to the postinfarct heart at 3 days and 2 weeks, with ≈14.5% of injected GFP (green fluorescent protein)-Muse cells estimated to be engrafted into the heart at 3 days. The migration and homing of the Muse cells was confirmed pharmacologically (S1PR2 [sphingosine monophosphate receptor 2]-specific antagonist JTE-013 coinjection) and genetically (S1PR2-siRNA [small interfering ribonucleic acid]-introduced Muse cells) to be mediated through the S1P (sphingosine monophosphate)-S1PR2 axis. They spontaneously differentiated into cells positive for cardiac markers, such as cardiac troponin-I, sarcomeric α-actinin, and connexin-43, and vascular markers. GCaMP3 (GFP-based Ca calmodulin probe)-labeled Muse cells that engrafted into the ischemic region exhibited increased GCaMP3 fluorescence during systole and decreased fluorescence during diastole. Infarct size was reduced by ≈52%, and the ejection fraction was increased by ≈38% compared with vehicle injection at 2 months, ≈2.5 and ≈2.1 times higher, respectively, than that induced by mesenchymal stem cells. These effects were partially attenuated by the administration of GATA4-gene-silenced Muse cells. Muse cell allografts and xenografts efficiently engrafted and recovered functions, and allografts remained in the tissue and sustained functional recovery for up to 6 months without immunosuppression. CONCLUSIONS: Muse cells may provide reparative effects and robust functional recovery and may, thus, provide a novel strategy for the treatment of acute myocardial infarction.

Truncated forms of U2 snRNA (U2-tfs) are shunted toward a novel uridylylation pathway that differs from the degradation pathway for U1-tfs.

During the biogenesis of U1 small nuclear ribonucleoprotein, a small population of U1 snRNA molecules acquires an extra methylation at the first transcribed nucleotide and a nucleolytic cleavage to remove the 3' structured region including the Sm protein-binding site and stem-loop 4. These modifications occur before hypermethylation of the monomethylated 5' cap, whereby producing truncated forms of U1 snRNA (U1-tfs) that are diverted from the normal pathway to a processing body-associated degradation pathway. Here, we demonstrate that a small population of U2 snRNA molecules receives post-transcriptional modifications similar to those of U1 to yield U2-tfs. Like U1-tfs, U2-tfs molecules were produced from transcripts of the U2 snRNA gene having all cis-elements or lacking the 3' box. Unlike U1-tfs, however, a portion of U2-tfs received additional uridylylation of up to 5 nucleotides in length at position 87 (designated as U2-tfs-polyU) and formed an Sm protein-binding site-like structure that was stabilized by the small nuclear ribonucleoprotein SmB/B' probably as a part of heptameric Sm core complex that associates to the RNA. Both U2-tfs and U2-tfs-polyU were degraded by a nuclease distinct from the canonical Dis3L2 by a process catalyzed by terminal uridylyltransferase 7. Collectively, our data suggest that U2 snRNA biogenesis is regulated, at least in part, by a novel degradation pathway to ensure that defective U2 molecules are not incorporated into the spliceosome.