In the study of cellular RNA chemistry, a major thrust of research focused upon sequence determinations for decades. Structures of snRNAs (4.5S RNA I (Alu), U1, U2, U3, U4, U5, and U6) were determined at Baylor College of Medicine, Houston, Tex, in an earlier time of pregenomic era. They show novel modifications including base methylation, sugar methylation, 5′-cap structures (types 0–III) and sequence heterogeneity. This work offered an exciting problem of posttranscriptional modification and underwent numerous significant advances through technological revolutions during pregenomic, genomic, and postgenomic eras. Presently, snRNA research is making progresses involved in enzymology of snRNA modifications, molecular evolution, mechanism of spliceosome assembly, chemical mechanism of intron removal, high-order structure of snRNA in spliceosome, and pathology of splicing. These works are destined to reach final pathway of work “Function and Structure of Spliceosome” in addition to exciting new exploitation of other noncoding RNAs in all aspects of regulatory functions. A key element in the study of cellular RNA metabolism is the molecular characterization of RNA. This characterization requires accurate determination of the RNA sequence. It is imperative to understand how RNA structure complements the functional definition of RNA. Cellular RNAs are posttranscriptionally modified at various points in the primary RNA transcript as well as processed. In cellular RNA metabolisms, RNA maturation is performed through various structural alterations that include chemical modifications of constituent components. A most representative modification is observed in chain shortening, rearrangements by transfer of phosphodiester linkages involved in splicing mechanisms (pre-mRNA), deletions (pre-rRNA), and transsplicing . Another is chain expansion demonstrated by modifications observed on polyadenylation, U-addition at 3′ ends, 5′-cap formation at 5′ ends, and insertions within trypanosome RNA. Other examples of modifications are base modifications, such as deaminations, methylations, hypermodifications, and ribose methylations.32.2.7G caps occurred within the last 50 years. They also contain their own specific modified nucleotides such as Ψ, m6A, m2G, and 2′-O-methylated nucleotides  + 28S  RNA in eukaryotes. The mRNAs contain the least modified nucleotides, with the exception of the 5′ end cap structure and occasional mIn ensuing years, massive scale DNA sequencing was advanced to accommodate the “Human Genome Project.” Two groups published the genomic map where the coding genes were cataloged. It was conservatively estimated that there are 25,000 genes and 50,000 proteomes involved in cell metabolism. It was also envisioned that processing mechanisms could be discerned by comparing the genomic structure with the RNA sequence determined using cDNA methods. Based on the ever-increasing number of RNA sequences, it was determined that most coding RNAs mature as a result of alternative splicing. Aberrant splicing is attributed to point mutations in the genetic code and splicing code -γ-ATP by polynucleotide kinase [32P]-5′-pCp by RNA ligase -derivative method can be useful. The radioactive labeling of terminals was performed using the periodate oxidation method, followed by reduction with [3H]-borohydride. T2 RNase digestion and fractionation by paper chromatography reveal the presence of the 3′-terminal and 5′-cap.In some cases, a rapid diagnostic examination is required. Most convenient procedures employ the use of specific antibodies against different forms of 5′-cap structure (mRNA can be labeled in vivo (prelabeling) or in vitro (postlabeling).32P]-phosphate in a phosphate-free medium. RNA is uniformly labeled by this method.In vivo labeling is carried out by incubation of living cells in the presence of [32P]-phosphate or [3H]-borohydride. [32P]-labeling can be carried out using kinase enzymes. The 5′-labeling is done with [32P]-ATP by polynucleotide kinase, that is, provided the 5′-end is free from phosphate. If the 5′-end is blocked by the presence of a 5′-cap structure, the pyrophosphate moiety must be removed by a pyrophosphatase and phosphatase. And then the kinase method can be employed to introduce the tracer. Labeling at the 3′-end is done with [32P]-pCp by RNA ligase. The [3H]-derivative (nucleotide diol) with [3H]-borohydride indicates that the 3′-end is free from phosphate or any other blocking structures. A shortcoming of [32P]-labeling is the short half-life of the isotope which provides a working period of approximately 4 half-lives. The main limitation of the [3H]-labeling method is weak energy of the tritium isotope. This can make the reading of the autoradiograph for a ladder sequencing gel very difficult.In vitro labeling is called postlabeling because it labels the isolated RNA with isotopic agents such as -RNA is hydrolyzed, and its products are separated by 2-dimensional paper chromatography followed by autoradiography [RNase Tiography . Since tiography .3H]-derivatives and separated by thin layer chromatography. The separated nucleosides  are detected by fluorography and identified based upon a standard migration pattern  or high voltage electrophoresis on cellulose acetate at pH 3.5 followed by DEAE homochromatography at 60–70°C. Another method that can be used is two-dimensional thin layer (PEI) chromatography using two-solvent systems . DetectiSeveral enzymatic digestions can be exploited.32P]-oligonucleotides (prelabeled) are subjected to secondary digestions with RNase U2 for placement of A residues, RNase T1 for G residues, RNase A for U, and C residues plus other endonucleases. Treatment with exonucleases , and partial digestion with the enzymes above is required to sequence RNA. In each step, nucleotide composition is determined.The recovered [A mobility shift test can be applied . After p32P]-oligonucleotides are applicable. Secondary digestion methods and accompanying [3H]-derivative methods for the determination of nucleotide composition can be carried out.The procedures used for prelabeled -KBH4) reduction. The reaction was carried out in 0.1 M sodium acetate buffer at pH 5 with freshly prepared NaIO4 in the dark for 1 hour and precipitated the RNA with ethanol. The RNA was redissolved in the same buffer and treated with ethylene glycol to destroy excess NaIO4. The RNA was precipitated with ethanol and redissolved in 0.1 M sodium phosphate buffer, pH 7.7, and treated with radioactive [3H]-KBH4 -KBH4 at pH 6 for 2 hours in the dark to produce trialcohol derivatives of nucleosides. All nucleosides with base modifications, except 2′-O-ribose modified, were labeled with tritium. The tritium-labeled trialcohol derivatives were separated by two-dimensional TLC (thin layer chromatography) on cellulose thin layers . Twenty milliliter (packed volume) of cells was incubated with 500 mCi of [32P]-orthophosphate in 1 liter of medium  for 9–16 hours  by NaIO4 and [3H]-KBH4, digested with RNase A, showed enzyme-resistant oligonucleotide eluting close to the pentanucleotide region on a DEAE column  [strongly basic cation exchange (quaternary amine)]. As shown in 3H]-labeled U1 RNA 5′ oligonucleotide, following digestion with snake venom phosphodiesterase and alkaline phosphatase, was separated by chromatographic methods with standards. Two-dimensional TLC (thin layer chromatography) and paper chromatography demonstrated that the [3H] labeled compound is a trimethylguanosine derivative , the cap core was treated with NaIOreaction . m32,2,7GpppAmpUmpApCp. The product was chromatographed on a DEAE column with standard AMP, ADP, and ATP. The product was eluted close to ATP, indicating that it is pppAm. This experiment proved that the 5′ oligonucleotide structure is 4 and [3H]-KBH4 was digested with RNase A. The labeled oligonucleotide eluted around the tetranucleotide region  was separated by HPLC. The composition was Am, Um, C, and mctively,  12]. Th. Th32,2id phase . 32,2,7G were typical of a trimethyl G nucleotide . The remaining oligonucleotide did not have m32,2,7G, indicating that the m32,2,7G is linked by pyrophosphate linkage pApGpCp. From these data, obtained by UV, [m32,2,7GpppAmpUmpApCp. The oligonucleotide sequence was deduced as in the case of U1 RNA. The structure is identical to the U1 5′ oligonucleotide 5′ Trimethylguanosine cap for the snRNA,5′ 7-mehtylguanosine cap for the mRNA,5′ 2,7 dimethylguanosine cap of virus and nematode RNAs5′ mpppG of U6 RNA.See .(pppNp) for primary transcripts such as 4.5S RNA I, 5S RNA, and Alu RNA. (pNp) 5′ end for processed RNAs such as Alu RNA, 5S RNA, tRNA, YRNA.3′2′-O-methylated; 4.5S RNA III3′ poly-A; mRNA, lncRNA3′ poly-U; polymerase III transcripts such as 4.5S RNA I, 5S RNA, and others3′ CCA; tRNA, U2 RNA.32,2,7G capping reactions are very interesting because trimethylguanosine is found only in noncoding RNA cap structures, although some nematode mRNA species also contain m32,2,7G caps. The snRNAs are less abundant (105 copies) than ribosomal RNA or tRNA (106 copies). Isolating large amount of RNA can be a hurdle to overcome. Massive preparative procedures and syntheses were pivotal for the thorough analysis of these modifications. The 2′-O-modifications occur mostly internally, and 3′ Um was also found in 4.5S RNA III. The RNA ribose with 2′-O-methylation confers resistance to enzymatic digestion by such enzymes as RNase A, RNase T1, RNase U2, and RNase T2 . They are also resistant to alkaline hydrolysis, and the alkaline hydrolysates can be separated into di-, tri-, and tetranucleotides by column chromatography and then by two-dimensional paper chromatography , and trimming is performed by 5′ → 3′ exonuclease Xrn1 and Rat 1 [Initially, the mn U3 RNA . Since t,7G caps , 88. In nd Rat 1 .32,2,7G cap, and U6 has the mpppG cap instead. They are present in complexes as RNP with proteins specific for each RNA as well as some common snRNP proteins such as the Sm proteins. Functionally, U1 RNP acts at 5′ splice sites and U2 RNA at branch sites including 3′ splice sites. U4, U5, and U6 snRNAs enter the spliceosomal intermediate as a tri-snRNP complex.These include U1, U2, U4, U5, and U6 snRNAs. All of these except U6 contain the m32,2,7G cap formation. Not all Cajal bodies contain the hTR, and it may be a transient localization for the maturation of hTR in the Cajal body. In the absence of Tgs1, the telomere of yeast S. cerevisiae has elongated single-stranded 3′ overhangs and TLC1 (1200 nt telomerase RNA) lacks the m32,2,7G cap. The absence of Tgs1 causes premature aging of yeast [Human telomerase RNA has a structure containing the H/ACA motif with 8 conserved regions (CR 1–8) . The CR7of yeast , 94.C. elegans has mRNA with the m7G cap as well as m32,2,7G cap, and the expression is regulated differentially. The genes for protein coding are monocistronic as well as polycistronic, and introns are much smaller than observed in mammalian cells. The polycistronic genes contain 2–8 operonic genes regulated by the same promoters. Some gene products are not processed, and others are spliced by cis-splicing as well as transsplicing. The transsplicings are carried out by SL RNA 1 or SL RNA 2. The approximately 110 SL RNA 1 genes are in tandem in chromosome V. The SL RNA 2 is derived from SL RNA 1 and there are ~18 dispersed genes with a variety of variant SL2 RNAs . They are all 100–110 nucleotide long and contain m32,2,7G caps and Sm protein binding sites. These pre-mRNAs, containing 5′ outron (monocistronic and 5′ first gene in polycistronic operonic genes), are transspliced by SL RNA 1 and internal operonic pre-mRNAs are mostly transspliced by SL RNA 2 and these genes have typically U-rich sequence containing ~100 bp spacers between two cleavage sites. The internal mRNA gene of polycistronic operonic genes, lacking a spacer, is transspliced always by SL RNA I [32,2,7G cap containing 22 nucleotides of SL RNA at their 5′ ends. The SL RNA (splice leader RNA) has a m32,2,7G cap and Sm protein binding sites. The nematode C. elegans has 5 eIF4E isoforms of cap binding proteins. They are IFE-1 , IFE-2 , IFE-3 (m7G cap binding only), IFE-4 (m7G cap binding only), and IFE-5 . The homolog amino acids W56 and W102 stacking the m7G caps in mice eIF4E are W51 and W97 in IFE-3 and W28 and W74 in IFE-5 . These include U1, U2, U4, and U5 spliceosomal RNAs, and U3, U8, and U13 nucleolar RNAs. Recently, telomerase RNA (S. cerevisiae TLC1) has also been reported to have a trimethylguanosine cap structure. The trimethyl-G caps are formed on cap 0 or cap I of m7G caps of pre-snRNAs by dimethylation of N2 position by trimethylguanosine synthase (Tgs1). The Tgs1 has been found to be in the Cajal body and cytoplasm. The U3 snoRNA is hypermethylated in the Cajal body, and U1, U2, U4, and U5 snRNA have been reported to be hypermethylated in the cytoplasm. Trimethylguanosine cap synthesis is carried out by multiple steps involving modifications. Trimethyl G caps are present in snRNAs involved in splicing and also in snoRNA involved in rRNA processing and modifications such as Ψ formation (H/ACA snoRNA) or 2The RNA polymerase initiates the RNA transcription with 5′ triphosphate nucleotides and in a majority with purine nucleotides of ATP or GTP. The capping reaction in a polymerase II system occurs cotranscriptionally within the nascent transcript of ~30–50 nucleotides. The guanylyltransferase is attached to heptad (YSPTSPS) repeats of CTD of RNA polymerase II. It was reported with cloned mouse guanylyltransferase and synthetic heptad repeats that the serine 5 phosphorylated 6 heptad repeats stimulated guanylyltransferase activity 4-fold. Serine 2 phosphorylation also binds the guanylyltransferase but did not stimulate enzyme activity . The capThe enzymes involved are RNA triphophosphatase and RNA guanylyltransferase, which can be found in the same enzyme, catalyze removal of one phosphate from pppNp initiation nucleotide, and transfer GMP from GTP through intermediary GMP-lysine phosphamide enzyme complex. The RNA guanyl 7 methyltransferase methylates the guanine at N7 position. The RNA 2′-O-methyltransferase methylates penultimate nucleotide 2′ OH, producing the cap 1 structure. In rat liver, it has been reported that 2′-O-methylation may precede the guanosine N7 methylation . The capping reactions by mammalian and shrimp capping complexes   have beeThe monomer of the 69–73 kDa protein has functions of RNA triphosphatase and RNA guanylyltransferase activity.NpNpNp-) , 100, anFrom HeLa cells, two enzymes forming cap I from cap 0 and cap II from cap I have been purified and characterized .n, m7GpppA(pA)n, m7GpppApGp, m7GpppApGpUp, and RNA with type 0 cap as substrates but not m7GpppA or GpppA. The substrate required for cap I formation should be at least a trinucleotide. This enzyme is present in both the nucleus (29.3 units/mg) and cytoplasm (3.74 units/mg) and cap II methyltransferase is exclusively in the cytoplasm (4.62 units/mg). Cap I methyltransferase uses GpppA(pA)The order of 7-methylation of ultimate G nucleotide and 2′-O-methylation of penultimate nucleotide is uncertain, and both pathways may occur.7G cap and 3′ polyadenylation is then transported into the cytoplasm as a complex with CBC20/80, PHAX, and Crm1-RanGTP. The m7G cap binds to CBC20 (156 amino acids) in complex with CBC80 (790 amino acids). The crystal structure of the CBC20/80 complex in association with m7G cap has been reported [7G cap is sandwiched between Tyr 43 and Tyr 20. And Phe 83, Phe 85, and Asp 116 have essential role for m7G cap binding. Asp 116 and Trp 115 interact with the N2 amino group and confer specificity of the m7G cap for other structures , the CRM1  or exportin 1 and RanGTP (Ras-related nuclear antigen). The snRNPs in the cytoplasm are trimethylated and processed. The mature RNA is reimported into the nucleus in a complex with the trimethyl G cap-specific binding protein snurportin 1 and snRNA binding proteins of Sm RNP and SMN proteins.The snRNAs synthesized by RNA polymerase II with mDespite immunofluorescent staining of U1 and U2 RNA exclusively in the nucleus , biochem7G cap, has been shown to be hypermethylated in HeLa cell cytoplasmic extracts and Sm binding site in U1 RNA is required [7G cap has been shown to be hypermethylated into the m32,2,7G cap structure in enucleated xenopus oocytes [7G capped unprocessed U1 RNA is retained in the nucleolus and splicing becomes cold temperature sensitive. The same enzyme is responsible for the U3 nucleolar RNA hypermethylation [The U1 snRNA  and U2 srequired . The Tgs oocytes . In yeas oocytes . In the hylation . The conhylation , 106. Thhylation .7G and 3′ extension of 10–15 nucleotides, is detected in Cajal bodies. The nucleolar localization requires the CAB box, hypermethylation to m32,2,7G cap, and maturation of the 3′ end [In HeLa cells, transfected U3 RNA gene products are trimethylated and mature U3 RNA is localized in the nucleolus. Immature U3 RNA, with both me 3′ end . Unlike e 3′ end , 108.The Tgs1, trimethylguanosine synthase in human, protein is 110 kDa and 852 amino acids in chain length. The gene is located in chromosome 8q11. The mRNA is 3.2 kb in length and produces a 110 kDa protein and ~65–70 kDa protein that is proteasome processed. The long form is in the cytoplasm, and the short isoform has been reported to be localized in the Cajal body within the nucleus. The Tgs1 has S-AdoMet methyltransferase signature motifs of X, I, II (include post 1 motif), III, IV, V, and VI , 110. The human Tgs1 motifs are the following. EGWFSVTPEKIAEHI/FA(a.a.682),motif X is (a.a.665)DRmotif I is (a.a.693)VVVDAFCGVGGN(a.a.704),VIAIDIDPV/IKI(a.a.725) and post 1 motif is VIAID which is responsible for S-AdoMet binding to the enzyme, motif II is (a.a.714)Rmotif III is (a.a.740)KIEFICGDFLLLAS(a.a.753),PWGGPDYA(a.a.771/772),motif IV is (a.a.758/759)VVFLSPmotif V is (a.a.785/786)DGFEIFRLSK(a.a.794/795), motif VI is (a.a.798/799)NNIVYFLPRNADI(a.a.810/811),π stacking with m7G guanosine of the substrate. The motif 1 and post 1 motif are reported to interact with S-AdoMet. [It was reported that trimethylation catalytic activity is located in the C-terminal region (amino acids 631–852) and this region contains the S-AdoMet-dependent methyltransferase motifs. The tryptophan in motif 4 is involved in -AdoMet. . The C-t-AdoMet. .γ and RXRα [The N-terminal portion of the molecule (amino acids 1–~477) has been reported to contain GXXGXXI, a K-homology domain for RNA binding, and a motif for SmB and SmD1 binding. The Tgs1 has also been shown to interact with PRIP (proliferator-activated receptor-interacting protein), and the N-terminal portion (amino acids 1–384) of Tgs1 has been shown to have stimulatory effects on transcription of PPARand RXRα , 110.α-helices and 7 β-strands. It is composed of 2 domains, the core domain (Glu675-Asp844) and N-terminal extension (Leu34-Ser671) connected by 3 amino acids—Val672, Thr673, and Ser674. The core domain consists of 7-β-strands in topology of   β6↑β7 ↓ β5↑β4↑β1↑β2↑β3↑ with a classical class 1 methyltransferase fold resembling the Rossmann-fold AdoMet-dependent methyltransferase superfamily [α-helices form a separate small globular subdomain involved in recognition and binding of both substrates. The residues Glu667 and Phe670 in motif X as well as Pro765, Trp 766, and Pro769 in motif IV are in proximity permitting the top of their binding clefts to be close together. Tryptophan 766 and m7G are stacked in a coplanar manner with a 3.2 Å distance providing a tight π-π interaction between them (The human Tgs1 (618–853) has been crystallized for structural analysis. The one monomer consists of 11 erfamily . The N-teen them . n2 substitution reaction. The N2 of m7G does the nucleophilic attack on an activated methyl group of the AdoMet  dissociate from the enzyme. Tgs1 can use m22,7G as a substrate, and newly bound AdoMet can methylate at the same position by the same mechanism to form the m32,2,7G cap structure. Dimethylation is not processive. After formation of mIn Drosophila melanogaster, DTL (Drosophila TAT-like) has been reported to exhibit trimethylguanosine cap formation activity for both U2 and U4 snRNAs. The mRNA for the protein Tgs1 is polycistronic and 2,600-nucleotide long with upper and downstream ORFs (open reading frames). The uORF is 80 bp from the transcription start site and has coding capacity for a 178 amino acid protein while dORF is 538 bp from the 5′ end and produces a 60 kDa protein (491 amino acids). The two cistrons are overlapped by 76 bp. Mutational analysis indicates that both the uORF and dORF regions are required for viability. The putative product of uORF contains periodic Leu residues, but there is no evidence that this region is translated at any time during Drosophila development. The protein from dORF contains an Arg-rich motif KKKRRQRQI similar to the RNA binding motif RKKRRQRRR in HIV TAT. This protein is localized in the nucleus and responsible for trimethylation of U2 and U4 snRNAs .S. cerevisiae, Tgs1 is in the nucleolus and U3 RNA is also in the nucleolus. In the absence of Tgs1, the pre-U3 RNA was found within the nucleolar body and U1 RNA was retained in the nucleolus. S. cevisiae, S. pombe, and Giardia lamblia Tgs1 can methylate m7GTP, m7GDP, and m7GpppA as substrates without preassembly of snRNP containing Sm proteins. The Tgs1 of S pombe is 239-amino-acid long and m7G is the pre-requisite for this reaction [In yeast reaction .lamblia has 2 enzymes, Tgs1 and Tgs2. Tgs 1 is not a processive enzyme but distributive and produces m32,2,7G in excess of AdoMet and enzyme. However, Tgs2 produces only m22.7G, and some G. lamblia RNAs contain dimethylG caps. The G lamblia Tgs1 has 300 amino acids and Tgs2 is 258 amino acids long. They all have landmark motifs for Ado-Met-dependent methyltransferase motifs [The e motifs .Trypanosoma brucei SL RNA (splicing leader) has the biggest 5′ oligonucleotide, type IV, of m7Gpppm26,6AmpAmpCmpm3UmpAp [26,6A and m3Um methylating enzymes have not been identified as yet [The parasite pm3UmpAp , 115. EnTrypanosoma brucei triphosphatase, 253 amino acids), TbCe1 , and TbCmt1 . The second is a set of fused enzymes possessing dual activities. It is TbCgm1 (Trypanosoma brucei cap guanylyltransferase and methyltransferase 1) that has 1050 amino acids [7G methyltransferase from T. cruzi and L. major [There exist two enzyme systems for 5′ cap formation. The first is the system composed of separate independent enzymes which are TbCet1 , and U4 snRNAs were reported to be synthesized by RNA polymerase III in Trypanosomes.They contain a Kpe 1 cap . The KDK26,6A, m3U base methylations, and fourth nucleotide 2′-O-methylating enzymes are not known yet.The TbMTr2 and TbMTr3 are responsible for second and third nucleotides 2′-O-methylations. The enzymes that perform mβ binding site at its N-terminus (amino acids 1–65) and trimethyl G cap binding site at amino acids 95–300 forming a cap binding pocket. This protein has more resemblance to mRNA guanylyltransferase. The snurportin 1 binds the trimethyl G cap forming π-stacking with tryptophan 276 and the penultimate purine nucleotide G  which is not suited for mass spectrometry. It is convenient to probe chemical complexity based on mass. The detailed analysis may require an unpredictably large amount of samples. There are 135 modified nucleosides listed, among which 6 nucleosides are not thoroughly identified [In the process of oligonucleotides cataloging, it is natural that an examination of base composition will reveal modified nucleotides or nucleosides in addition to unmodified standard nucleotides or nucleosides. In routine work, identification of modifications can be readily made by two-dimensional paper chromatography for nucleotides or thin layer chromatography for nucleosides. However, there may be an occasion where chromatographic identification is not sufficient. Of course, it is best to have collaboration with outside specialists. For the sake of structural microanalysis, it is highly recommendable to determine molecular weight of the unknown nucleotide or nucleoside by mass spectrometry . The reqPast sequence work has permeated numerous significant areas of research providing a better understanding of cellular metabolism. The information obtained thus far is RNA-based information which is not seen in DNA, proteins, and others. As sequence work continues to make enormous progress, the postgenomic era will shape the direction of research in the area of molecular mechanisms of RNA metabolism. They are briefly as follows.In RNA maturation, knowledge of structural modifications is necessary to discern between various mechanistic options. For example, there are two molecular mechanisms mediated by catalysis. One is mediated by RNA enzymes (snRNAs and snoRNAs) involved in splicing of pre-mRNA and processing of pre-rRNA. The other is protein enzymes involved in 5′ cap formation. Currently, the higher order structural analysis is in progress. There is a need to elucidate the details of molecular mechanisms. Along with the study of splicing physiology, splicing pathology is making significant progress. Aberrant modifications can generate disease causing alterations in structure. The aberrations cause problems in reading both genetic codes and splicing codes. Studying the regulation of alternative splicing will clarify the selective rules in intron removal and pathogenic rules in splicing code. From these studies, corrective strategy will evolve. The present sequence work is engaged in definition of ncRNAs diversity and their functional roles . Since imiRNA: MicroRNA (imperfect base pairing)siRNA: Small interfering RNA (perfect base pairing) tasiRNA: Transacting small interfering RNA natsiRNA: Natural antisense transcribed small interfering RNApiRNA: PIWI interacting RNA (RNA precipitated by PIWI protein antibody) rasi RNA/pitRNA: Repeat-associated small interfering RNA/pi-target RNAPARs: promoter associated RNAs PROMTs: Promoter upstream transcripts (sense and antisense transcript)PASRs: Promoter-associated small RNAsTSSa-RNAs: Transcription-start-site-associated RNAs tiRNAs: Transcription initiation RNAsMSY-RNA: MSY2-associated RNAs snoRNA: Small nucleolar RNA sdRNA: sno-derived RNAsmoRNA: MicroRNA-offset RNAstel-sRNA: Telomere small RNAs crasiRNA: Centrosome-associated small interfering RNAs hsRNA: Heterochromatin small RNA or hairpin small RNA scaRNAs: Small Cajal-body-associated RNAsY RNAs: Cytoplasmic small RNA Y1, Y3, Y4, and Y5tRNA-derived RNAs: Small RNA processed from tRNA by RNase (angiogenin)Alu/SINE RNA: Alu restriction enzyme cleaved repeat gene transcript/short interspersed nucleotide element RNA.TR/TERC: Telomerase RNA/telomerase RNA componentNEAT RNAs: Nuclear enriched abundant transcript 1 RNAs NEAT1v-1: NEAT1 variant 1 NEAT1v-2: NEAT1 variant 2 NEAT2/MALAT1: Metastasis associated in lung adenocarcinoma transcript 1PINC RNA: Pregnancy-induced noncoding RNADD3/PCA3: Prostate-cancer-associated RNA 3PCGEM1: Prostate cancer gene expression marker 1SPRY4-1T1: Sprouty homolog 4 gene transcript 1 (melanoma specific).xiRNAs: X chromosome inactivating RNAsXist: X chromosome inactivating sense RNATsix: Antisense transcript of Xist RepA: Repeat A RNAAIR RNA: Igf2r imprinting region RNAH19: Igf2 imprinting region RNAKCNQ1ot1: Antisense RNA from intron 10 of Kcng1 gene imprinting region.HOTAIR: Homeogene inactivating RNABORG: BMP/OP-responsive-gene-associated RNACTN RNA: Cationic amino acid transporter protein coding region RNAANRIL RNA: Antisense noncoding RNA in INK4 locus.LINE: long interspersed nucleotide element CSR-RNA: Immunoglobulin class switch recombination region RNA. Mud: Mutant U1 dieRES complex: Heterotrimeric RNA retention and splicing complex composed of Bud13, Ist3/Snu17, and Pml1Swt21: Synthetic with Tgs1 number 21TRAMP complex : Interacts with exosome in the nucleus and involved in 3′ end processing of rRNA, snoRNA, and U1, U4, and U5 snRNA;  Trf4 or Trf5: poly(A) polymerase(PAP);Mtr4: RNA helicase;Air1 or Air2: Zinc knuckle proteinCbf5(YLR175W): Centromere binding factor; Pseudouridine synthase catalytic subunit of box H/ACA snoRNP complexPIMT: PRIP-interacting protein with methyltransferase domain, PIMT is a Tgs1 (trimethylguanosine synthase 1) cloned from human liver cDNA libraryPRIP: PPAR interacting proteinPPAR: Peroxisome proliferator-activated receptorPBP: PPAR binding protein.


Marsilea vestita dehydrate as they mature. During this process both mRNA and proteins required for subsequent development are stored within the microspores as they become fully desiccated and enter into senescence. At this point microspores become transcriptionally silent and remain so upon rehydration and for the remainder of spermatogenesis. Transcriptional silencing coupled with the translation of preformed RNA makes the microspore of M. vestita a useful system in which to study post-transcriptional regulation of RNA.Many rapidly developing systems rely on the regulated translation of stored transcripts for the formation of new proteins essential for morphogenesis. The microspores of the water fern Marsilea homolog of the Exon Junction Complex core component Mago nashi.We have characterized the distribution of mRNA as well as several conserved markers of subnuclear bodies within the nuclei of desiccating spores. During this period, nuclear speckles containing RNA were seen to aggregate forming a single large coalescence. We found that aggregated speckles contain several masked mRNA species known to be essential for spermatogenesis. During spermatogenesis masked mRNA and associated speckle proteins were shown to fragment and asymmetrically localize to spermatogenous but not sterile cells. This asymmetric localization was disrupted by RNAi knockdown of the M. vestita. The asymmetric distribution of specific mRNAs to spermatogenous but not sterile cells mirrors their translational activities and appears to require the EJC or EJC components. This suggests a novel role for nuclear speckles in the post-transcriptional regulation of transcripts.A subset of masked mRNA is stored in association with nuclear speckles during the dormant phase of microspore development in  M. vestita is an aquatic, heterosporous water fern whose sporophyte resembles a four-leaf clover. Its microspores and megaspores are meiotic products that desiccate and become dormant after they are formed. Upon rehydration, the microspores develop rapidly to produce male gametophytes that make multiciliated spermatozoids [for review, [ review, .M. vestita depends on little or no new transcription. The microspore becomes transcriptionally silent during its desiccation and remains so upon rehydration and initiation of spermatogenesis [Like other rapidly developing systems -7, male ogenesis . Therefoogenesis . In thisM. vestita [Not surprisingly, the translation of specific stored transcripts is under tight temporal and spatial control -12. One  vestita . Centrin vestita ,9,10. ThAn important mechanism regulating gametophyte development is the unmasking of stored transcripts for translation [for review, . Within Recently, we found that the polyamine, spermidine (SPD), acts as a temporal regulator for releasing the masked, stored transcripts in the gametophyte ,15. ExogNuclear speckles are small aggregations (~1 μm) of 20-25 nm granules that occupy the interchromatin space of many eukaryotic nuclei . SeveralM. vestita is a transcriptionally silent system that relies on the translation of stored mRNA after the spore is released from desiccation, and contains a subset of nuclear localized masked transcripts, we suspected that examining nuclear speckle dynamics might lead to insights into developmental control in the maturing gametophyte.Stress conditions such as hypoxia, inhibition of respiration, transcription, phosphorylation and ethanol treatment have been shown to cause the sequestration of the Exon Junction Complex (EJC) core component eIF4A-III to several subnuclear structures including nuclear speckles ,32. It iin situ hybridization (FISH), in an assay designed to distinguish between masked and unmasked (qc-mRNA) populations of the same transcripts in fixed cells. We demonstrate the utility of this assay by tracking the movements of specific transcripts initially stored in association with nuclear speckles into the cytoplasm of the antheridial mother cell. We show subsequent movements of masked transcripts into spermatogenous, but not jacket cells of the developing gametophyte, and that this asymmetry may be regulated via the EJC component Mv-Mago. The asymmetric distribution of one of these transcripts (centrin) mirrors its pattern of translation and presents a likely mechanism for post-transcriptional regulation essential for cell fate determination during the rapid and precise process of spermatogenesis in M. vestita.In this study, we have examined nuclear speckle dynamics during microspore entry into dormancy and transcriptional quiescence. We show that in addition to cytoplasmic stores, aggregated nuclear speckles serve as sites of poly(A)+ RNA storage. We developed a novel variation on fluorescence We examined nuclear speckle dynamics during microspore desiccation, since this is the developmental period when the gametophyte becomes transcriptionally silent. We harvested microspores from green, mostly-submerged sporophytes prior to the beginning of dehydration and subsequently every 2 weeks thereafter, as the ponds containing the sporophytes were allowed to dry out.For all experiments using sectioned material presented here, thousands of microspores were grown, fixed, and embedded. Each experiment was conducted independently a minimum of 2 times. Between 30-40 sections were used per slide so that many hundreds of sectioned spores were observed for each experimental trial, of which a no less then 20 representative photographs were taken to ensure accurate analysis. The number of independent trails per experiment, and total number of photographs taken for each set of experiments are listed in Additional file SC35 is a non-snRNP splicing factor that is often used as a specific marker of nuclear speckles [for review, . We usedWe used the metachromatic dye Toluidine Blue O  in the desiccating microspore, which we expected to label large nucleoli. This rRNA was present as a conspicuous nuclear particle Figure , and exhRehydration of the microspore triggers the commencement of spermatogenesis, but does not disrupt transcriptional silencing. We were interested in seeing if speckles remained aggregated after microspore rehydration. We examined the distribution of nuclear speckle markers, SC35 and U2B", a snRNP splicing factor known to be present in nuclear speckles. Since a subset of poly(A)+ RNA is retained in speckles during transcriptional inhibition , we usedM. vestita. In ISH assays, 5S and 25S rRNA were not detected within the nuclei but were abundant throughout the cytoplasm of newly hydrated microspores  probe to detect poly(A)+ RNA movements during development. At 30 minutes of development, a large aggregate of poly(A)+ RNA was seen within the nucleoplasm of undivided microspores Figure . At the isolates .Since precociously unmasked RNAs are detectable within the nucleus of the newly hydrated spore after 10 mM polyamine additions , we reasTo confirm that masked transcripts are associated with aggregated speckles, and then, to examine their role in spermatogenesis, it was essential to find a method for visualizing specific masked transcripts in unperturbed microspores. We found that traditional ISH and FISH methods work well for determining the localization of qc-mRNA transcripts, but these strategies fail to label masked transcripts ,15 prese2b, eWe made short 25mer biotinylated DNA probes complementary to SPDS and centrin mRNAs, transcripts that become detectable by ISH within the nucleus after treatments of spores with 10 mM SPD . The 25mDual FISH labeling with a poly(T) probe and a 25mer SPDS probe showed that foci of specific masked transcripts localize with nuclear speckle aggregate-associated poly(A)+ RNA Figure . Double We were interested in what happens to nuclear speckle components as they exit the nucleus, so we tracked the abundance and distribution of specific transcripts at various time points during spermatogenesis, starting as the gametophytes progressed through their first division cycle Figure . Using sBy the end of all nine mitotic division cycles in the gametophyte, small foci of both SPDS and centrin masked transcripts were apparent in the cytosol, adjacent to the nucleus of each spermatid Figure . These fSimilar to masked mRNA, U2B" protein exhibited localized immunolabeling in aggregates within the antheridial initials Figure . By 4 hoM. vestita [Previously, the RNAi-induced silencing of Mv-Mago, a homolog of the EJC component Mago nashi, was shown to deplete Mv-Mago levels within the spore as well as disrupt the endogenous asymmetry between spermatogenous and sterile cells in the developing male gametophyte of  vestita . A prima vestita , where c vestita .Since Mv-Mago may also play a role in the asymmetric distribution of nuclear speckle components, including masked centrin mRNA, the effect of Mv-Mago silencing on the distribution of both masked mRNA and associated proteins was assessed. RNAi has previously been demonstrated to abolish detectable translation of Mv-Mago . GametopM. vestita relies on the regulated translation of stored mRNA for the rapid production of motile spermatozoids. The packaging of these transcripts for long-term storage in the spore is an essential mechanism required for rapid development leading to male gamete formation. In this paper, we have shown that nuclear speckles enlarge and aggregate as transcription is silenced in the desiccating microspore. In cells with blocked transcription, nuclear speckles often enlarge and serve as sites of storage for pre-mRNA processing machinery and under these conditions, a subset of poly(A)+ RNA is retained within the nucleus associated with nuclear speckles [The male gametophyte of speckles .M. vestita.Transcription in the microspore remains silent even upon hydration and recovery from dormancy. We have shown that during spermatogenesis, nuclear speckles are maintained as a single coalescence in the nucleoplasm of the newly hydrated microspore. Morphologically, the nuclear speckle aggregate superficially resembles a nucleolus. However, the lack of colocalization with traditional nucleolar markers (Fibrillarin and rRNA) in the subnuclear aggregate reduces the likelihood of this possibility. Moreover, our studies of the desiccating microspore demonstrate a marked loss of nucleolar markers during microspore entry into quiescence. Like many dormant systems that stockpile polysomes -40, our M. vestita, no CBs examined have been shown to be associated with poly(A)+ RNA [for review, [U2B", which is present in the subnuclear aggregation, is commonly used as a marker of Cajal bodies (CBs) in both animals and plants. Is the aggregate a CB? Coilin is widely considered to be a diagnostic marker of the CB in animals ,42, wher review, . In addi review, . Using tM. vestita resembles traditional nuclear speckles more closely than other kinds of nuclear inclusions. Typically, nuclear speckles consist of small, interchromatin aggregations of pre-mRNA splicing proteins, but have been shown to enlarge upon transcriptional inhibition [M. vestita and traditional speckles are inescapable. Beyond the storage of pre-mRNA processing machinery, nuclear speckles could serve as sites for the storage of mRNA during periods of inhibited transcription and/or splicing. Intriguingly, a subset of speckle associated poly(A)+ RNA is masked mRNA (SPDS and centrin) that is known to be essential for gametophyte development in M. vestita. It is important to point out that with the required use of Proteinase K in our in situ hybridization protocols, FISH and immunofluorescence labeling had to be performed sequentially as described in the Methods section. Immunofluorescence labeling had to be performed first, followed by imaging and removal of coverslips, prior to treating the cells for FISH. We attempted a number of methods that would allow simultaneous immuno- and in situ labeling on the same sections of gametophytes, but we had to resort to sequential labeling in order to obtain reproducible patterns of antibody and RNA distributions in the cells. The sequential method used has the unfortunate drawback that in between imaging sections can become distorted because of the harsh incubation conditions for FISH or ISH. We feel that this is why in all such experiments + RNA. In addition to similarities in composition, the behavior of the aggregated nuclear material in the microspore of hibition . This enhibition . Our obss Figure  subnucles Figure  or nucles Figure .Nuclear speckles remain aggregated within the nucleus of the microspore until the first division. During the first division, both protein and RNA associated with nuclear speckles enter the cytosol adjacent to the nucleus of the antheridial initial. As additional division cycles progress, this material is asymmetrically distributed to spermatogenous cells, but not to jacket cells. During spermatogenesis, foci of masked transcripts were visible in the cytoplasm directly adjacent to the nuclei of spermatogenous cells, but were not seen in jacket cells. Sectioned material was used for the analysis of this asymmetric masked transcript distribution, because FISH and immunofluorescence assays cannot be performed together on intact, but fixed spores, even if they are imaged by confocal microscopy. Thus, while the foci of masked centrin and SPDS RNAs should not be expected to be visible in all of the spermatogenous cells in a single section of a single gametophyte, observations of many sections of many gametophytes provide strong indications that all of the spermatogenous cells contain these foci of masked transcripts.While the ISH and FISH assays carried out in this study were not directed against RNA contained exclusively within jacket cells, previous studies (15) have demonstrated that the techniques used here are capable of labeling transcripts contained exclusively within jacket cells. In addition Figure There are two critical co-variants in gametophyte development: a) the patterns of divisions leading to the formation of sterile and spermatogenous cells are precise inside the spore wall, and b) there is no cell movement during development. These factors highlight an essential process in cell fate determination in the gametophyte: the fragmentation of aggregated nuclear speckle-associated masked transcripts must be orderly to ensure that appropriate complements of mRNAs are distributed among all of the spermatids. The movements of masked transcripts must be under strict control during the successive division cycles, so that appropriate parcels of transcripts are ultimately allocated among the 32 spermatids in the gametophyte. Thus, masked mRNA, originally stored in a single coalescence of nuclear speckles within the nucleus of the desiccated microspore, is dispersed as a set of masked RNA-containing particles that are uniformly distributed among spermatogenous cells, but not to sterile jacket cells, which arise from the same progenitors.M. vestita [The EJC exerts profound effects on the symmetry of divisions in the developing gametophyte of  vestita . By sileArabidopsis [M. vestita where mRNA is stored during desiccation in association with nuclear speckles. We propose that the association of this RNA with these speckle components ensures nuclear retention that in turn, forestalls its translation.In other plants, the EJC and in particular Mago nashi have been shown to be essential for development in nearly all tissues and organs. The silencing of atMago in bidopsis  results bidopsis ,32 in nubidopsis . The prebidopsis ,32. We bin situ hybridization probes later becomes detectable with these long probes in the cytoplasm of spermatogenous cells at time points when the corresponding proteins become abundant [in vitro translation assays [Mv-Mago silencing results in the symmetric distribution of masked centrin mRNA as well as the symmetric translation of centrin in both spermatogenous and jacket cells at 4 hours of development. Masked centrin mRNA initially stored in the nucleus of the microspore is clearly required for centrin protein production in the spermatogenous cells of the gametophyte. It follows that additional levels of translational regulation must be in place to inhibit the translation of cytoplasmic centrin mRNA, which is ubiquitously present in all cells at all times during spermatogenesis . Althougabundant . When con assays  that shoM. vestita. We believe this coalescence contributes substantively to the long-term storage of stable, masked RNAs that may be present as fully or partially processed transcripts, which are essential for the formation of spermatozoids within hours after the dry microspore is hydrated. Furthermore, it is apparent that the mode of storage of these transcripts may play a role in their translational regulation and their asymmetric distributions during spermatogenesis.The discovery of a coalesced nuclear speckle aggregate, whose function includes the storage and masking of developmentally important transcripts, reveals an important level of post-transcriptional regulation affecting rapid development of spermatozoids in Paraspeckles, subnuclear aggregates closely associated with nuclear speckles  retain CSpisula oocytes enters the cytoplasm following activation, and centrosomes form within the diffusing nucleolinus. In parthenogenetically activated oocytes, laser ablation of the nucleolinus results in a failed meiotic division and microtubule disorganization, demonstrating a clear functional role for the nucleolinus in spindle formation and cell division [Many subnuclear bodies (including nuclear speckles) have a cytoplasmic phase in their cycles ,16,52 anOngoing and future characterizations of the subnuclear masked mRNA and associated processing machinery should provide greater insights into regulatory mechanisms that underlie this rapid developmental process. We believe that traditional 'long' ISH or FISH probes could not detect masked mRNA because masking agents (most likely proteins) obscure sites of hybridization. We believe because of their small size our 25mer probes can be fit in between their masking agents and thus robustly label masked mRNA. We would like to identify these masking agents and further assess their role(s) in development and RNA regulation. An obvious area of interest centers on the fragmentation process and the distribution of masked transcripts among the spermatids. Since both centrin and SPDS masked transcripts become localized exclusively in foci within spermatogenous cells, these transcripts can reveal patterns of storage, movement and unmasking for particular mRNAs during development. Our FISH assays show that transcript pools may be stored discretely, so how and when are these particles fragmented and passed on to spermatogenous cells? If masked transcripts are stored in multiple foci, how does each spermatid receive a full complement of masked transcripts? How is the timing of translation controlled for specific transcripts during development? The mechanisms that control how the subnuclear RNA and pre-mRNA processing machinery becomes asymmetrically distributed between spermatogenous and sterile cells remains unclear. Positioning of the subnuclear material in the cytoplasm during the prothallial division clearly underlies this process, and identifying the components and factors that affect movements of these molecules will be important in understanding cell fate determination mechanisms in the highly ordered gametophyte.M. vestita. In addition, we show that both protein and nucleic acid components of this speckle aggregate are asymmetrically localized to spermatogenous cells during development. This localization requires the EJC core component Mago nashi. Asymmetric localization of masked mRNA mirrors both the temporal and spatial patterns of their corresponding proteins. We believe this suggests a role for nuclear speckles in the post-transcriptional regulation of mRNA.We show here that translationally masked RNA is stored within aggregated nuclear speckles during desiccation and dormancy in the microspore of Marsilea vestita microspores were obtained, cultured, fixed, and sectioned as previously described [escribed ,9.All widefield fluorescence microscopy was performed with a Zeiss Axioscope equipped with standard Fluorescein, TexasRed and UV filter sets. Confocal microscopy was performed on a Zeiss LSM700 using Zen 2009 software. Subsequently, .lsm stacks were exported into ImageJ 1.44k and rendered as 3D models using the ImageJ 3D Viewer plugin.DAPI and pyronin Y are available from a number of commercial sources. DAPI was diluted in PBS and used at a final concentration of 2.5 μg/ml. Pyronin Y was used at 5% in deionized water. Pyronin Y (5%) was added 1:1 with 0.2M pH 4 acetate buffer on the day of use to make PY staining solution. Slides with sectioned material were incubated in acetone for 15 minutes on a rocker. Slides were next incubated in PBS for five minutes; this was repeated twice. Samples were placed in a humid chamber and incubated with DAPI for 10 minutes followed by incubation in PBS-T for five minutes and then PBS for five minutes on a rocker. Slides were incubated in a humid chamber for 1.5 minutes with PY staining solution, rinsed with deionized water and dehydrated in an ethanol series . Slides were dipped briefly in xylene.http://www.vectorlabs.com/). Biotinylated poly(T) probe was purchased from GibcoBRL. 25mer biotinylated DNA probes for the detection of masked centrin and SPDS transcripts were obtained through the custom DNA oligo service at Integrated DNA Technologies http://www.idtdna.com.Probes for 5S and 25S rRNA were constructed through digoxigenin-11-dUTP incorporation as previously described ,13. SingThe ISH protocol used here has been previously described . DetectiFISH on masked transcripts was performed as detailed previously  with theet al. [http://www.abcam.com) and used at a dilution of 1:200. Goat-anti-biotin, AlexaFluor 594 conjugated goat-anti-mouse and AlexaFluor 594 conjugated rabbit-anti-goat antibodies were obtained from Molecular Probes  and were used at 1:200, 1:1000, and 1:1000 dilutions respectively. Histochemical detection of antibodies was carried out using an alkaline phosphatase conjugated anti-mouse antibody, followed by visualization with nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.Toluidine Blue O staining  was perfet al. . AntibodIn double labeling experiments, antibody labeling was performed as described above, coverglasses were mounted but not sealed. Following imaging, coverglasses were removed by incubating slides in PBS-T, 3 times for 5 minutes on a rocker. Following coverglass removal, our standard FISH protocol was followed as detailed above.RNA interference was performed as described by Klink and Wolniak . Briefly10 mM Spermidine treatments were performed as previously described .TCB performed experiments, participated in the design of experiments and interpretation of the data, and writing of the manuscript. SMW participated in the design of experiments, the interpretation of data, and the writing of the manuscript. TCB and SMW both read and approved the final manuscript.Table S1. Numbers of independent trials and representative photographs for experiments presented. Column 1 lists the experiment(s). Column 2 lists the figure(s) with representative images from specific experiment(s). Column 3 lists the total number of photographs taken for each experiment(s). Column 4 lists the total number of independent trials for each experiment(s).Click here for fileFigure S1. Differential fluorescent labeling of DNA and RNA via dual DAPI/PY staining. Microspores fixed at 30 minutes of development. PY (green)  and DAPI (blue)  detected via 488 nm and UV illumination respectively. a-b, Microspore stained with PY (green). c-d, Microspore stained with DAPI (blue). e-f, Microspore double-stained with DAPI (blue) and PY(green). g-h, Microspore pretreated with RNase and double-stained with both DAPI (blue) and PY (green). i-j, DNase pretreated samples were double-stained with DAPI (blue) and PY (green). k-l, DNase and RNase treated samples were double-stained with DAPI (blue) and PY (green). Bar = 25 μm.Click here for fileMovie S1. Pyronin Y labeling reveals discreet aggregations of subnuclear RNA after 2 weeks of dehydration. Microspores collected and fixed after 2 weeks without watering were sectioned and double stained with DAPI and PY. Subnuclear PY (green) signal was detected in successive confocal slices and these were rendered as a 3D model. Movie is in .mov format playable with QuickTime.Click here for fileMovie S2. Pyronin Y labeling reveals a partial coalescence of RNA aggregates after 4 weeks of dehydration. Microspores collected and fixed after 4 weeks without watering were sectioned and double stained with DAPI and PY. Subnuclear PY (green) signal was detected in successive confocal slices and these were rendered as a 3D model. Movie is in .mov format playable with QuickTime.Click here for fileMovie S3. Pyronin Y labeling reveals total coalescence of RNA aggregates after 6 weeks of dehydration. Microspores collected and fixed after 6 weeks without watering were sectioned and double stained with DAPI and PY. Subnuclear PY (green) signal was detected in successive confocal slices and these were rendered as a 3D model. Movie is in .mov format playable with QuickTime.Click here for fileFigure S2. RNA is not detectable within the nuclei of microspores after the first division. a-c, Microspore fixed and sectioned after 1.5 hours of development. a, DAPI (blue). b, Pyronin Y (green). c, merge of a and b. Bar = 25 μm.Click here for fileMovie S4. Spermidine additions cause the partial dissociation of aggregated nuclear RNA. Microspores were incubated with 10 mM SPD for 4 hours, fixed, sectioned and double stained with DAPI and PY. Subnuclear PY (green) and DAPI (blue) signals were detected in successive confocal slices and these were rendered as a 3D model. Movie is in .mov format playable with QuickTime.Click here for fileMovie S5. Spermidine additions cause the total dissociation of aggregated nuclear RNA. Microspores were incubated with 10 mM SPD for 4 hours, fixed, sectioned and double stained with DAPI and PY. Subnuclear PY (green) and DAPI (blue) signals were detected in successive confocal slices and these were rendered as a 3D model. Movie is in .mov format playable with QuickTime.Click here for fileMovie S6. 'Short' FISH probes detect foci of subnuclear masked transcripts that are distinct from chromatin. FISH against masked SPDS (red) was conducted on 20 μm sections taken from microspores fixed after 30 minutes after hydration. Probes were detected using avidin bound TexasRex. Sections were counterstained with DAPI (blue). Movie is in .mov format playable with QuickTime.Click here for fileFigure S3. Traditional FISH probes fail to detect masked SPDS transcripts within the nuclei of maturing microspores. a-c, Traditional biotinylated probes directed against SPDS transcript (red). Pre-prothallial (a), mid-prothallial (b), and late-prothallial (C) division microspore. The prothallial nucleus denoted by "p" in b and c. Bar = 5 μm.Click here for fileFigure S4. Defects in asymmetric division are not requisite for symmetric distribution of subnuclear material in Mv-Mago knockdowns. a-c, microspore subjected to Mv-Mago RNAi and fixed after 5 hours of development. (a) DAPI (blue), (b) phase contrast, (c) 4G3 labeling of U2B" (red). d-f, representative microspore subjected to Mv-Mago RNAi and fixed after 4 hours of development. (d) DAPI (blue), (e) phase contrast, (f) masked centrin transcripts (red) detected with 25mer biotinylated FISH probes. Spermatogenous cells denoted by "sp" and jacket cells denoted by "jk." Bar = 25 μm.Click here for file


The fate of an mRNA is largely determined by its interactions with RNA binding proteins (RBPs). Post-transcriptional processing, RNA stability, localisation and translation are some of the events regulated by the plethora of RBPs present within cells. Mutations in various RBPs cause several diseases of the central nervous system, including frontotemporal lobar degeneration, amyotrophic lateral sclerosis and fragile X syndrome. Here we review the studies that integrated UV-induced cross-linked immunoprecipitation (CLIP) with other genome-wide methods to comprehensively characterise the function of diverse RBPs in the brain. We discuss the technical challenges of these studies and review the strategies that can be used to reliably identify the RNAs bound and regulated by an RBP. We conclude by highlighting how CLIP and related techniques have been instrumental in addressing the role of RBPs in neurologic diseases. This article is part of a Special Issue entitled: RNA and splicing regulation in neurodegeneration. As an mRNA progresses through the various regulatory stages, from transcription to translation and degradation, it is associated with a myriad of multifunctional RNA-binding proteins (RBPs). Indeed, recent studies suggest that hundreds of RBPs interact with mRNAs to collectively determine their fate . For exaA crucial challenge of researchers studying the functions of RBPs is to identify the RNAs regulated by a specific RBP. CLIP enables study of RBP–RNA interactions that occur in intact cells or tissues since the biological samples are UV cross-linked, forming a covalent bond between the RNAs in direct contact with the RBP. The RBP-of-interest is then immuno precipitated, and the co-purified RNA is ligated to adapters that enable production of cDNA libraries for sequencing  Fig. 1AAFig. 1A.A common expectation from the CLIP studies is that they will directly generate a shortlist of the functionally relevant RNA targets, enabling the study of pathological relevance of these RNAs and the development of new RNA-based therapies. CLIP is clearly a step towards this goal. However, each RBP can regulate a great multitude of RNAs by diverse mechanisms which presents a greater challenge than appreciated at first sight. The frequency of CLIP sequence reads that identify a specific site depends not only on the affinity of the RBP for this site, but also on the RNA abundance. It is clear that RNA abundance varies between genes, and can vary also within each gene due to variable kinetics of transcription, splicing and intronic RNA degradation. It remains challenging to adequately account for these factors in data analysis. The tendency to report the absolute number of RNA targets should thus be approached with caution, since these numbers depend on the variable quantitative quality of CLIP cDNA libraries and require the use of arbitrary binding thresholds. In appreciation of these points, we describe several approaches that can be used to increase the confidence in assigning functionally relevant RNA targets of RBPs.The original CLIP method , which wThe challenge to identify functionally relevant RBP targets of an RBP is particularly great for RBPs that bind to pre-mRNAs in the nucleus. Due to the great length of pre-mRNAs, a binding site for an RBP is present in most expressed pre-mRNAs. If sequencing is carried out to sufficient depth, CLIP studies typically identify multiple binding sites of each RBP in most expressed pre-mRNAs. It is therefore crucial that CLIP data are quantitative, in order to identify discrete regions where high cross-linking density corresponds to high-affinity binding. iCLIP was developed to preserve the quantitative information by using primers for reverse transcription that contain a random barcode. Analysis of this random sequence enables computational filtering of high-throughput sequencing data to remove the artefacts caused by variable PCR amplification of different sequences .An alternative method to increase the efficiency of CLIP employs photoactivatable ribonucleoside enhanced cross-linking and immuno-precipitation (PAR-CLIP) . HoweverA crucial step in the analysis of CLIP data is to identify sites where RBPs bind with high-affinity, which can be identified by clusters of reads (in CLIP) or clusters of cross-link events (in iCLIP or PAR-CLIP). This in turn acts to reduce the prediction of false positive RNA targets. The most common approach used in the past studies is to calculate the probability that a cluster of reads that is present in a specific genomic region (such as an intron or a gene) is statistically significant when real CLIP data are compared to randomised CLIP data within the same region . This apTo normalise mRNA abundance for RBPs that bind to mRNAs, mRNA sequencing (RNAseq) should be carried out simultaneously with CLIP experiments, and used to normalise the number of CLIP reads by the RNAseq reads  Fig. 1BBFig. 1B.Evolutionary conservation of the sequence motifs bound by the protein can support the functional importance of the RNA binding site C. For inGenome-wide studies have revealed that RBPs often control diverse aspects of post-transcriptional regulation. Initial biochemical studies were based on a small number of regulated RNAs, therefore RBPs that regulate splicing were often classified as splicing repressors or enhancers. Studies of Nova showed that it can enhance or repress exon inclusion in many different RNA targets , and thiIntegration of CLIP data with analysis of global changes of splicing has been used to identify the global positional principles governing the splicing functions of Nova . The comThe integrative analysis has three main advantages. Firstly, the RNA map can validate the high quality of data by demonstrating that binding position correlates with the effect of the protein (enhanced vs. repressed), and that binding at these positions in the regulated RNAs is significantly enriched relative to the control RNAs. Secondly, since the RNA map defines the positions where RBPs bind to have functional impact, it enables separation of those changes observed in RNAseq data that are the result of direct effects of the RBP from those that may result from indirect effects. This is particularly important for studies in the brain, since manipulation of RBPs can lead to developmental defects, cell stress, changes in neuronal networks or glia–neuron interactions, siRNA off-targets, and other effects that can indirectly change gene expression. Thirdly, the positional pattern in the RNA map can reveal new regulatory mechanisms employed by the RBPs. For instance, RNA maps demonstrated that the enhancer elements are most often located immediately downstream of exons. Moreover, they showed that certain RBPs can also regulate splicing via distal binding sites .To date, RNA binding profiles of several RBPs involved in neurologic disorders have been investigated either in cell culture, transgenic animal models, or human post-mortem tissue . Here weAgrn and Dab1 leads to defects in the formation of the neuromuscular junction and in neuronal migration in the CNS of the Nova1−/−/Nova2−/− mice, respectively  is responsible for fragile-X syndrome, the most common inherited form of mental retardation. FMRP is an important regulator of translation . A studyDMPK) (ZNF9) (Muscleblind-like proteins (Mbnl1 and Mbnl2) are sequestered in various tissues within the neuromuscular diseases of myotonic dystrophy types 1 (DM1) and 2 (DM2) in response to expansions of CUG repeats in the 3′UTR of the myotonic dystrophy protein kinase (DMPK) , or CCUG) (ZNF9) , respect) (ZNF9) . The RNA) (ZNF9)  was exte) (ZNF9) . It willALS and FTLD are two diseases with phenotypic and mechanistic overlap . FamiliaAnother observation made by FUS CLIP was a saw-tooth binding pattern along long introns, similar to that seen in total RNAseq . HoweverhnRNPC leads to widespread expression of cryptic exons, which can result in decreased gene expression. In the absence of hnRNP C, cryptic exons are recognised by U2AF65, a splicing factor that initiates the recognition of 3′ splice sites  disease models . InducedOver the next years, we can expect more CLIP studies to assess RNA interactions of multiple RBPs at a specific stage of brain development, which will enable comprehensive comparative studies. It will be possible at that point to define the phenomena specific to RBPs involved in neurodegenerative disorders, and separate these from the more general aspects of protein–RNA interactions.


Overfishing threatens coral reefs worldwide, yet there is no reliable estimate on the number of reef fishers globally. We address this data gap by quantifying the number of reef fishers on a global scale, using two approaches - the first estimates reef fishers as a proportion of the total number of marine fishers in a country, based on the ratio of reef-related to total marine fish landed values. The second estimates reef fishers as a function of coral reef area, rural coastal population, and fishing pressure. In total, we find that there are 6 million reef fishers in 99 reef countries and territories worldwide, of which at least 25% are reef gleaners. Our estimates are an improvement over most existing fisher population statistics, which tend to omit accounting for gleaners and reef fishers. Our results suggest that slightly over a quarter of the world’s small-scale fishers fish on coral reefs, and half of all coral reef fishers are in Southeast Asia. Coral reefs evidently support the socio-economic well-being of numerous coastal communities. By quantifying the number of people who are employed as reef fishers, we provide decision-makers with an important input into planning for sustainable coral reef fisheries at the appropriate scale. How many fishers do coral reefs support? This question is important to fisheries managers and coastal planners throughout coral reef regions. Coral reefs support the nutritional and economic needs of fishers in many of the world’s poorest and most vulnerable communities We use the term coral reef fisheries to encompass fishing on coral reefs as well as associated habitats such as seagrass meadows, which often make up an important component of the reef fishing area Ensuring food security and adapting to climate change are among the primary challenges facing coastal nations today A number of studies have shown that fisher densities (number of fishers per area) affect the ecological state of coral reef fisheries. For instance, We compile a database of the number of coral reef fishers in all countries where coral reefs occur. Our estimate encompasses the number of coral reef fishers in the primary sector, i.e., those who catch fish, either full-time or part-time, for both artisanal and subsistence purposes. It also includes women and children who typically glean in shallow inshore areas for edible coral reef organisms. This global study provides an estimate of reef fisher numbers at the country level. We recognise that data at this scale may not be suitable for the development of community level interventions, which require finer scale studies that estimate undocumented fisher populations . Nevertheless, the estimates presented here provide a starting point for identifying potential countries where extra attention to finer scale studies is warranted.The socio-economic importance of coral reefs cannot be overstated. Annual net economic benefits derived from healthy coral reef ecosystems are estimated at around USD 30 billion a year, with reef fisheries contributing USD 5.7 billion of this Reef fisheries are intensely targeted not only for a local source of protein, but also for export oriented trades such as the live reef food fish, aquarium, and curio trades Around 100 countries and territories in the world have coral reefs , with InCoral reef fisheries are multispecies and multigear in nature. Fishing is usually undertaken by small-scale fishers who use unmotorised or low powered boats and manually operated gears such as hand lines, nets, traps, and spears. In many regions, destructive fishing methods such as the use of fish bombs and sodium cyanide are also widespread on coral reefs In 2006, approximately 3 million people worldwide, almost entirely from countries or territories with coral reefs, collected sea cucumbers We estimated the number of fishers in all coral reef countries, identified according to the UN Atlas of Coral Reefs We used two approaches for estimating the number of reef fishers, which we chose based on the availability of published literature, and on the following premise:Reef fishers constitute a fraction of a reef country’s total marine fishers. We assumed that this fraction is represented by the ratio of reef fish to total marine fish landed value of a country or territory;The number of reef fishers is a function of reef area, coastal population, and fishing pressure.We assumed that reef fishers are a subset of all marine fishers  in each reef country or territory. An estimate of the number of fishers worldwide is reported in Sea Around Us Project (SAUP) catch database (www.seaaroundus.org), except for Florida, which was not reported separately in the database. We identified 200 reef fish species and taxon groups in the SAUP catch database  by the reef area of each country or territory. Fisher density in each country was defined by two variables: i) rural coastal population size; and ii) fishing pressure:We calculated the number of reef fishers by multiplying fisher density  and fishing pressure (pf);i.ia) was based on estimates reported in the UN Coral Reef Atlas iD) for each reef country or territory was defined by rural coastal population and fishing pressure exerted on their respective coral reefs. Previous studies have shown a correlation between human population levels and fishing effort in coral reef areas Reef area  to be ‘low’; population levels between the 50–75th percentile were considered to be ‘medium’, while those above the 75th percentile were ‘high’.Rural coastal population data were extracted from the Socioeconomic Data and Applications Center’s (SEDAC) Global Rural-Urban Mapping Project . We used global assessments rather than going by a case by case basis in order to minimise the variability in assessment methods across nations and territories. The general analytical approach used in these publications is briefly summarised below:The level of fishing pressure was determined based on mainly qualitative assessments derived from 3 primary publications: Reefs at Risk Series Reefs at Risk Series: These studies used GIS (Geographic Information System) to map where reefs were at risk from degradation due to local threats, including overfishing, and global threats, such as climate change. The threat to coral reefs from overfishing was evaluated based on coastal population data, extent of fishing areas, occurrences of destructive fishing, and presence of marine protected areas. The level of threat was also informed by input from coral reef scientists and experts. The risk levels were low, medium, high, and very high;Status of Coral Reefs in the World: These reports relied on the expert opinions of local reef scientists and managers from around the world to determine the status of each nation’s coral reefs, and the extent to which reefs were threatened by human activities such as overfishing;Newton et al. (2007): Unlike the above 2 studies which assessed the level of threat to coral reefs from overfishing, this study evaluated the status of 49 reef fisheries worldwide. Each reef fishery was categorised as being underexploited, overexploited, or fully exploited, based on estimates of how much national reef fish landings exceeded maximum sustainable yield from coral reefs.Each country was categorised as having high fishing pressure if its reef fishery was assessed to be overexploited in −2 of fishing ground) corresponding to inshore small scale fisheries were assigned to nations that were now segregated by rural coastal population and fishing pressure. For instance, American Samoa was assigned a fisher density level of 8.6 fishers⋅km−2  of this range to represent high, low, and medium fisher densities  to represent high, low, and medium fisher densities. We applied the same procedure of eliminating extreme endpoint values in the 6 Pacific island case studies. We then took the averaged maximum, minimum, and midpoint fisher density values across all 6 cases to represent high, low, and medium fisher densities  in low rural coastal population countries  because gleaners were already included in the base estimate from Countries or territories were considered to have substantial gleaning if they were mainly subsistence based or if fishing was part of their cultural identity. Based on these criteria, all island nations in the Western Pacific were determined to have substantial gleaning. We chose a 1∶1 gleaner to coral reef fisher ratio because gleaning is usually done by women and children for subsistence purposes. Therefore, we conservatively assumed that in places where gleaning is widespread, each fishing household had at least one other member besides the fisher who gleaned.We further identified countries where some gleaning took place, but not at as high a rate as in the Pacific islands. These countries were extracted from ad hoc search of the literature for data or indicators relating to coral reef fisher numbers  to determine whether our estimates were within similar orders of magnitude. More systematically, we calculated the proportion of coral reef fishers to rural coastal population in each country or territory. It was reported by The final number of coral reef fishers in 99 countries (excluding Singapore) was then calculated as the average of approach 1  and 2 (fisher density), with the results from each approach defining the upper and lower range. To verify whether our estimate was within reasonable bounds, we conducted an We used 70% as a cut-off for all countries and territories except for small island nations in the Western Pacific, where nearly all households in coastal villages are involved in fishing Approach 1: The average percentage of reef related to total marine fish landed value across all 99 countries and territories was 0.28. We applied this to the total number of fishers of each country or territory to derive the number of coral reef fishers. We also tested a maximum and minimum of 50% and 10%, given that the highest and lowest regional average reef/total marine landed value percentages were 43% and 11%, respectively.−2). We also tested how results changed if all reef fisheries were considered to be i) underexploited (fisher density of 5.2 fishers⋅km−2); and ii) overexploited (fisher density of 21.5 fishers⋅km−2), using the fisher densities corresponding to medium population levels.Approach 2: We multiplied the coral reef area for each country or territory by the overall average fisher density across all countries and territories  to 6.8 million . The estimated 6 million reef fishers represent about 2% of the 275 million people who live within 30 km of reefs around the world A recent study estimated 3 million sea cucumber fishers worldwide In total, fisher estimates for 11 countries exceeded the 70% rural population limit, and these were replaced with estimates from alternate sources , although it exceeded the 70% cut-off. This is due to existing literature that reported about 116,000 people in Papua New Guinea’s artisanal fisheries For 2 cases (Guam and Papua New Guinea), only the fisher density approach was applied. For Guam, the standardised approach generated an estimate that exceeded the 70% cut-off. As we were unable to find an alternate source for Guam, we used the estimate generated by the fisher density method only, which did not exceed the 70% cut-off. The initial number of fishers estimated for Papua New Guinea (average of approach 1 and 2) did not exceed 70%.However, we chose to use the estimate provided by the fisher density method instead  also provides an opportunity for researchers to record the number of reef fishers in their surveys. Increasing focus on coral reef conservation can also be a means to record fisher numbers, as more non-governmental organisations initiate community conservation activities in coral reef dependent areas.Improving on our estimate will necessitate a concerted effort by reef countries and territories to start collecting data on the number of people fishing on coral reefs. This is not an insurmountable task – the Global Coral Reef Monitoring Network already provides a comprehensive review on the status of coral reefs every 4 years The fact that small-scale fishers account for 90% of fishers worldwide File S1Supporting tables. Table S1. Reef taxa used to identify reef catch from the Sea Around Us catch database. Table S2. Reef/total landed value % for reef countries in 2005. Table S3. Estimated number of reef fishers for reef countries and territories worldwide in 2010. Table S4. Comparison of fisher estimates from current study and alternate independent sources.(DOC)Click here for additional data file.


Priming of random hexamers in cDNA synthesis is known to show sequence bias, but in addition it has been suggested recently that mismatches in random hexamer priming could be a cause of mismatches between the original RNA fragment and observed sequence reads. To explore random hexamer mispriming as a potential source of these errors, we analyzed two independently generated RNA-seq datasets of synthetic ERCC spikes for which the reference is known. First strand cDNA synthesized by random hexamer priming on RNA showed consistent position and nucleotide-specific mismatch errors in the first seven nucleotides. The mismatch errors found in both datasets are consistent in distribution and thermodynamically stable mismatches are more common. This strongly indicates that RNA-DNA mispriming of specific random hexamers causes these errors. Due to their consistency and specificity, mispriming errors can have profound implications for downstream applications if not dealt with properly. RNA-seq is a widely used tool for transcriptome analysis and gene expression estimation. Most commonly, mRNA is fragmented followed by reverse transcription into first strand cDNA primed by random hexamers. Subsequently, second strand cDNA is synthesized from first strand cDNA by DNA polymerase, again initiated by random hexamer priming. Bias in hexamer distribution and CG content affects abundance estimates and several bias correction algorithms have been developed e.g. -5. Apart from bias in hexamer priming sites, random hexamer mispriming has recently been implicated in sequence read to reference mismatches. Because mismatches mainly occur in the first seven nucleotides of first strand cDNA ,6 and arThis paper deals with sequence read to reference mismatches commonly observed in RNA-sequencing data. Mismatches are defined as any position in sequencing reads that deviate from the reference to which these reads align. Mismatches can reflect 1) true biological variation, both in genomic DNA or caused by RNA-editing, or 2) errors in the library preparation process caused by hexamer mispriming or PCR errors, and finally 3) sequencing errors caused by the erroneous identification of bases in the sequencing process. We focus on errors that most likely arise in the library preparation phase. During RNA-seq library preparation, polyA+ RNA is fragmented and reverse transcribed into first strand cDNA initiated by random hexamer priming. We refer to mismatches between the reference fragment and the observed sequence that are caused by RNA-DNA hexamer mispriming during first strand cDNA synthesis as RD-mismatches. Similarly, we use the term DD-mismatches to describe errors caused by DNA-DNA hexamer mispriming during second strand cDNA synthesis.Errors caused by random hexamer mispriming have thus far received limited attention. While read trimming to exclude error-rich first stretches of RNA-seq reads is done in some studies ,11, therTaraxacum officinale RNA mixed with ERCC RNA spikes. Twenty-three libraries were multiplexed using Illumina’s multiplex sequencing assay and pooled with a 2% ERCC spike [We conducted a RNA-seq experiment of CC spike . SubsequCC spike ; GSM5170We analyzed two independently generated RNA-seq datasets, focusing on reads mapping to ERCC spikes which are artificial RNA fragments of known sequence that are added during library preparation. ReverseFirst strand cDNA mismatches in the first seven base pairs corresponding to the hexamer binding site and the base immediately downstream of this show position-dependent and nucleotide-dependent mismatch patterns . These sThus we conclude that mispriming is non-random and can be heavily biased, as RNA binding sites with a U at positions three and four (relative to the hexamers 5’ end) misprime with hexamers having a G at that position in ~88% of the mispriming cases. These specific mismatch patterns were observed consistently in two independent data sets . The disConsistent mismatch patterns observed in the first seven nucleotides of first strand cDNA will affect downstream applications such as de novo assembly, SNP calling and RNA-editing analysis. For instance, consistent and high (20%) mismatch rates can be problematic for k-mer assembly strategies, as these erroneous k-mers cannot be effectively combined with “true” k-mers. Because mispriming rates are not random and for some positions heavily biased  they canIdentification of the hexamers that are most commonly involved in mispriming as well as the type and position of the mismatches this generates can aid in the design of strategies to counter the effects that these errors can have in downstream applications. Our results suggest that it could be useful to explore less aggressive approaches than trimming. Such approaches could include post-mapping correction of 5’ mismatches, modifications to random hexamer design to exclude commonly mispriming hexamers and specific bias correction models that mask or remove the observed mismatches in the first seven bases of reads. Our analyses shows strong and consistent bias in sequence errors in 5’ ends of RNA-seq reads, which (1) strongly supports the hypothesis that random hexamer mispriming during first strand cDNA synthesis causes the errors, and (2) highlights the risk of errors in downstream applications as well as suboptimal data use. We conclude that technical artifacts in sequencing data are insufficiently described. Further research on random hexamer mispriming will inform optimized strategies to mitigate their negative effect on downstream analysis.


Classical swine fever virus (CSFV) is the member of the genus Pestivirus under the family Flaviviridae. The 5' untranslated region (UTR) of CSFV contains the IRES, which is a highly structured element that recruits the translation machinery. The 3' UTR is usually the recognition site of the viral replicase to initiate minus-strand RNA synthesis. Adenosine-uridine rich elements (ARE) are instability determinants present in the 3' UTR of short-lived mRNAs. However, the presence of AREs in the 3' UTR of CSFV conserved in all known strains has never been reported. This study inspects a possible role of the ARE in the 3' UTR of CSFV.in vitro and that the binding ability is conserved in strains irrespective of virulence.Using RNA pull-down and LC/MS/MS assays, this study identified at least 32 possible host factors derived from the cytoplasmic extracts of PK-15 cells that bind to the CSFV 3' UTR, one of which is HuR. HuR is known to bind the AREs and protect the mRNA from degradation. Using recombinant GST-HuR, this study demonstrates that HuR binds to the ARE present in the 3' UTR of CSFV This study identified one of the CSFV 3' UTR binding proteins HuR is specifically binding to in the ARE region. Classical swine fever virus (CSFV), along with Bovine viral diarrhea virus (BVDV) and Border disease virus (BDV), is a member of the genus Pestivirus under the family Flaviviridae. CSFV infects pigs and causes high economic losses especially in Europe and Southeast Asia. CSFV infection causes lymphopenia and thrombocytopenia, thereby evading and compromising the immune system , 1 μl of 100 mM Dithiotheratol, 20 units T7 RNA polymerase, 1 μg linearized template, 0.25 μl containing 25 mM each of ATP, CTP, and GTP, 0.25 μl containing 250 μM UTP, 3.5 μl of [α-32P]UTP containing a total of 11.65 pmole (35 μCi) and 20 units HPRI . The reaction was scaled up proportionally as required. Plasmids pT7-ALD3'UTR, pT7-LPC3'UTR, pT7-93KS 3'UTR and pT7-95TD 3'UTR DNA clones were linearized using SmaI for run-off transcription RNA labeling. The transcription reaction was performed at 37°C for 2 hrs and the samples were run on a 5% polyacrylamide gel. The appropriate bands were cut and the RNA was eluted from the gel. The 32P body-labeled RNA was checked for quality and then quantified.A typical 10 μl Binding was performed in a 10 μl reaction containing 1 μl of 10X binding buffer, 100 ng of yeast total RNAs, about 1 picomole of radio-labeled probe and 1 μg of protein  and 20 units HPRI. For competition assay equal mass (about 5 ng) of cold competitor RNA was used. For super shift assay, 1 μg of anti-HuR antibody was included in the reaction. The reaction was incubated for 10 min at room temperature and then run on a 5% native polyacrylamide gel. The gel was exposed to a phosphorimage plate for a few minutes and then scanned using PhosphorImager Fujifilm BAS 2500.2 dishes were washed with PBS, harvested and then washed in 5 volumes of hypotonic buffer and finally suspended in 3 volumes of hypotonic buffer and placed on ice for 10 min. Cells were lysed using a type B pestle Dounce homogenizer by using 10 to 20 strokes on ice. The lysate was centrifuged at 3300 g for 15 min at 4°C and the supernatant was saved as the cytoplasmic extract and its protein content estimated. The pellet contains nuclei.PK-15 (Porcine Kidney-15) cells were grown at 37°C in Eagle's minimal essential medium (EMEM) with 5% newborn calf serum (NCS) and 100 U/ml penicillin and 100 μg/ml streptomycin. Sub-cellular fractions were prepared as described . BrieflyIn order to be cross-linked, 25 μg of RNA was first oxidized in a 50 μl reaction containing 100 mM sodium acetate pH 5.0 and 5 mM sodium meta-periodate which was incubated for 1.5 hrs at RT in a shaker shielded from light. About 80 μl of agarose beads  were washed 4 times with and then re-suspended in 60 μl of 100 mM sodium acetate pH 5.0. Oxidized RNA was added to the agarose beads and incubated for 2.5 hr at RT. Then the RNA-agarose beads were washed thrice with 1 ml of 2 M NaCl then washed thrice in buffer D .About 60, 90, and 120 μg of PK-15 cytoplasmic extracts along with 10, 15, and 20 μg yeast total RNAs, respectively, were incubated for 5 min at RT, then added to RNA-agarose beads and placed in a shaker for 25 min at RT . The beads were spun down and washed four times in 1 ml of buffer D containing 125 mM NaCl. Then the beads were suspended in 10 μl 4× SDS-PAGE loading buffer and boiled for 5 min and loaded on to a 12% SDS-polyacrylamide gel and run for 65 min at 200V. The gel was stained with silver SNAP and bands of the indicated sizes were cut and sent for mass spectroscopy.E. coli BL21/DE3 cells transformed with pGEX-HuR. GST fusion protein was purified on glutathione-Sepharose beads as recommended by the manufacturer, and the concentration determined by comparison with a bovine serum albumin (BSA) curve in SDS-PAGE stained with Coomassie brilliant blue.For constructing the GST-HuR expression vectors, the coding region of HuR was amplified by PCR from human A431 cells cDNA using primers 5'TCTAATGGTTATGAAGACCACATG3' and 5'TTATTTGTGGGACTTGTTGG3', and cloned into pGEX-6P-1 vector (Amersham Bioscience). GST-HuR was prepared using A detailed analysis revealed five overlapping AUUUA pentamers. This pentamer is a typical adenosine and uridine-rich element (ARE), an instability determinant found in the 3' UTR of short-lived cellular mRNAs, which usually encode proteins required only for a short period, such as transcription factors, oncogenes, and cytokines . AREs arAgarose beads covalently cross-linked to CSFV 3' UTR RNA were used to pull down the proteins binding to the 3' UTR present in cytoplasmic extracts of PK-15 cells . The eluE. coli-purified GST-HuR was used in EMSA. Radioactive-labeled CSFV ALD 3' UTR RNA probe (about 1 picomole) interacts with GST-HuR, though not with BSA, even at a higher concentration  of polyA, polyC, polyG, and polyU were used as competitors , 93KS and 96TD (both moderately virulent Taiwanese isolates), and LPC (an avirulent vaccine strain).R Figure . The antR Figure . In reacWe have demonstrated that CSFV harbors bona fide AREs in the 3' UTR of its genomic RNA and that it is conserved in all analyzed strains. To the best of our knowledge, this is the first report showing that a viral genomic RNA of a positive-sense RNA animal virus harbors ARE motifs in its 3' UTR. The presence of an instability determinant such as the ARE in the genomic RNA of positive-sense RNA virus is seemingly suicidal. The presence of ARE in cellular mRNAs or certain RNAs of specific DNA viruses is understandable, though its presence in the sole RNA of CSFV appears harmful to its existence. However, since CSFV is extremely efficient in infectivity and multiplication and has a broad tissue tropism, the presence of the ARE hints that the virus could use the ARE interacting machinery of the host to its advantage.in vitro (Figures Using RNA agarose beads, we isolated host factors from PK-15 cytoplasmic extracts that interact with the CSFV 3' UTR, and identified many of them using LC/MS/MS. Data attained via LC/MS/MS indicate more than one AREBP; we chose to focus on HuR first because it has been reported to protect ARE containing mRNAs from degradation. The coverage of HuR is only 3%, which could be because HuR is a nuclear/cytoplasmic protein, though it is predominantly nuclear. This study has shown that purified recombinant GST-HuR binds to CSFV 3' UTR  Figures .Though HuR is predominantly nuclear, various types of stress, such as UV exposure, chemical stress, and heat shock, cause HuR to relocalize to the cytoplasm -29. WhilThe advent of low molecular weight inhibitors of HuR, which have demonstrated its drugability, could open exciting possibilities into the functional and mechanistic studies of HuR, and also in the therapeutic potential of combating viral infections . The rolIn conclusion, this study used the RNA pull-down and proteomics techniques to identify several host proteins interacting with the 3' UTR of CSFV RNA. One of the CSFV 3' UTR binding proteins, HuR specifically binds to the ARE region in the 3' UTR.The authors declare that they have no competing interests.MN prepared the HuR protein and did the EMSA analysis. MYC did the RNA pull down experiments. SWH prepare the labeled transcripts. CCH provided critical review of the manuscript. JTT and CHT were project supervisors, participated in the discussion of all experiments of the project and preparation of the manuscript.All authors read and approved the manuscript


FUS is a DNA/RNA binding protein found to be mutated in some cases of both sporadic and familial forms of ALS. It is still not clear how ALS-causing mutations in FUS leads to motor neuron degeneration. Here, we exploited a Drosophila model and mammalian neuronal cell lines to elucidate the role of the RNA-binding ability of FUS in regulating FUS-mediated toxicity. To determine the role of the RNA binding ability of FUS in ALS, we mutated FUS RNA binding sites  to leucines and generated RNA binding-incompetent mutants (4F-L) with and without ALS causing mutations R518K or R521C. We found that mutating 4F to L residues makes FUS RNA binding-incompetent. We observed that ectopic expression of RNA binding-incompetent FUS in fly brain, eyes and motor neurons strongly blocks neurodegenerative phenotypes as compared to RNA-binding-competent FUS carrying ALS causing mutations. Interestingly, RNA-binding deficient FUS strongly localized to the nucleus of Drosophila motor neurons and mammalian neuronal cells whereas FUS carrying ALS linked mutations was distributed to the nucleus and cytoplasm.in vivo unbiased genetic screen that led to the discovery of several dominant modifiers of mutant FUS toxicity including proteins involved in regulating SG dynamics, mitochondrial functions and RNA splicing. These genetic modifiers would help in identifying potential therapeutic targets for ALS.Importantly, we found that incorporation of mutant FUS into stress granules is dependent on the RNA-binding ability of FUS. SGs are dynamic aggregates composed of proteins and RNA that are formed when cells are under a variety of stresses. We observed that normally cytoplasmic SGs rapidly disassemble when stress conditions end, whereas cytoplasmic SGs formed in ALS patient cells having a FUS mutation fail to disassemble. This suggests that mutant FUS sequesters proteins and RNAs important for cellular homeostasis and the defect in disassembly of cytoplasmic SGs contributes to ALS. Finally, following up these observations we performed an