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Flipping through the first issue of RNA, two things specifically attracted my eyes: the original edition of The RNA World by Gesteland and Atkins, and a paper by Yi-Tao Yu et al. That classic monograph collected invaluable commentaries and reviews on the whole spectrum of RNA research from the legends and pioneers, on which I often relied for wisdom and insight. That paper by Tim Nilsen's group describing a phosphorothioate substitution study of U6 snRNA in nematode, and along with a similar study in yeast U6 by John Abelson's group, provided early chemical evidence that the bulging uridine (U69 in nematode and U80 in yeast) at the 3′ stem-loop of the U6 snRNA is likely an essential catalytic constituent of the spliceosome. Following on those observations, Shyue-Lee Yean, a postdoc in my lab, spent a good three to four years trying to find answers to a simple question: which comes first, the phosphorothioated U6 block or the Prp2 step? At that time Jean Beggs’s and my groups have shown that Prp2 is an RNA-dependent ATPase that is required for the onset of splicing in the assembled spliceosome. We knew that the ATP hydrolysis by Prp2 induces a conformational rearrangement of the spliceosome that proceeded catalysis. Would phosphorothioated U69/U80 inhibit the spliceosome activation step by Prp2? Shyue-Lee initially synthesized the U6 RNA using polymerase incorporation of the modified nucleotides, but the imprecision of the 3′ ends and low yields forced him to use ligase to stitch together chemically synthesized RNA fragments. Luckily with the help from John Termini's group we not only made the RP stereoisomers but also the SP isomers. The experiments not only answered our initial question—modified U6 does not block the Prp2 step—but also uncovered an unanticipated, magnesium coordinating, and catalytically relevant activity of U80SP. With recent elegant works by the groups of Joe Piccirilli and Jon Staley (in addition to the structural work by the groups of Sam Butcher and Dave Brow) we now know that U80 indeed participates in catalytic metal coordination along with additional nucleotides of U6, and that the spliceosome is indeed a ribozyme. As for Prp2, through the effort of the groups of Soo-Chen Cheng and Reinhard Luhrmann, among others, we now know that the ATP hydrolysis by Prp2 leads to reshuffle several proteins onto or off the spliceosome, explaining the “down-sizing” of the spliceosome by Prp2 that we observed almost two decades ago. A sequence between the branchpoint and the 3′ splice site is critical for productive Prp2 action; however, whether U6 snRNA is involved in this remains unclear. By working on yeast and human splicing factors, I found that not only human factors have more features but also the human cells have more complex responses to splicing perturbations. This is really nothing new; however, it reminds me of the conversation that I had with my musician daughter. She said that different music (classical, jazz, blues, etc.) has different building blocks as well as ways to arrange them; only with an understanding of that structure will a person be able to appreciate the meaning of that music (other than just a feeling). I wonder, although all biological systems have similar or conserved rules, some systems may predominantly use certain types of rules while others rely more on different ones. We have seen some intriguing examples for this in RNA biology—piRNA, RNA-dependent RNA polymerases, RNA-mediated transcriptional silencing, etc. Shall we be surprised if there are more—grand or subtle—to be found? In the midst of organizing and teaching an advanced graduate course on RNA, I found that over two-thirds of the course dealt with topics that were just or not even discovered when RNA was first published. Marvelously, those topics (microRNA, long non-coding RNA, CRISPR RNA, etc.) attract students to the RNA field and create a common language among colleagues from different fields. A legend and recombinant DNA guru in our institution had predicted that the past 50 years of DNA-focused research would give way to RNA-focused research. I am afraid that he is right, again. I would like to thank Tim Nilsen for the opportunity to share these few thoughts with the RNA readers. Although I cannot predict where RNA research is heading, I fully anticipate many new discoveries and excitement regarding RNA will continue in the next 20 years and beyond, so long as we remain curious and keep exploring (with generous supports from funding agencies).

PSF (PTB-associated splicing factor) is a multi-functional protein that participates in transcription and RNA processing. While phosphorylation of PSF has been shown to be important for some functions, the sites and the kinases involved are not well understood. Although PSF does not contain a typical RS domain, we report here that PSF is phosphorylated in vivo to generate an epitope(s) that can be recognized by a monoclonal antibody specific for phosphorylated RS motifs within SR proteins. PSF can be phosphorylated by human and yeast SR kinases in vivo and in vitro at an isolated RS motif within its N terminus. A functional consequence of SR phosphorylation of PSF is to inhibit its binding to the 3′ polypyrimidine tract of pre-mRNA. These results indicate that PSF is a substrate of SR kinases whose phosphorylation regulates its RNA binding capacity and ultimate biological function.

Laser pattern generators ALTA 3500 and 3700 are widely used for 0.18 micron and above technology nodes in photomask manufacturing. They have low butting, high throughput and high position accuracy, with some weaknesses such as, corner rounding, no proximity effect correction and poor CD linearity when compared to E-beam pattern generators. Optical Proximity Correction (OPC) software was thus created to extend the productivity of laser pattern generators. For contact holes serifs are typically added at the four corners to enhance pattern fidelity. However, the serifs or scattering bars can significantly increase the data size. In our study, we generated serifs for contact holes but applied different exposure strategies: (1) lumping serifs together with the main pattern; (2) exposing serifs and main pattern separately with same dosage; (3) exposing serifs and main pattern separately with different dosages. We examined the results of each approach in terms of contact hole quality, throughput, and inspection results.

The aim of this study was to investigate the fibroblast growth factor receptor 3 (FGFR3) mRNA cleavage by ribozymes targeting FGFR3, effect of growth inhibition and associated with mechanism on multiple myeloma (MM). We designated two ribozyme-expressing plasmids that target the FGFR3 genes, Rz52 and Rz32. In vitro catalytic activity of Rz52 and Rz32 in KMS11 cells decreased FGFR3 mRNA expression to 45% (p0.05) and 80% (p0.5), respectively, of that of the control. In vivo examination of the Rz52-transfected KMS11 clone showed that FGFR3 mRNA expression decreased to 20% (p0.05) of the control. In the Rz52-transfected H929 clone, FGFR3 mRNA decreased to 50% of the control. Protein expression of FGFR3 decreased to 70% of the parental KMS11 and H929 clones. DNA synthesis in the Rz52-transfected KMS11 clone decreased to 20% of that of the control, whereas the viability of cells decreased to 2% (p0.01) of that of the control. Ribozyme cleavage-associated increase in apoptosis of Rz52 KMS11 transfectants was twice that of the control. The inhibition of FGFR3 expression by ribozymes was associated with decreased vascular endothelial growth factor (VEGF) expression and upregulation of Flt-1 but not of the KDR receptor. Our data indicate that FGFR3 is an important cell survival and antiapoptotic factor for MM cells and that ribozyme-targeted downregulation of FGFR3 might be useful as a novel therapeutic intervention in MM characterized by t(4;14).

Prp2 is an RNA-dependent ATPase that activates the spliceosome before the first transesterification reaction of pre-mRNA splicing. Prp2 has extensive homology throughout the helicase domain characteristic of DEXD/H-box helicases and a conserved carboxyl-terminal domain also found in the spliceosomal helicases Prp16, Prp22, and Prp43. Despite the extensive homology shared by these helicases, each has a distinct, sequential role in splicing; thus, uncovering the determinants of specificity becomes crucial to the understanding of Prp2 and the other DEAH-splicing helicases. Mutations in an 11-mer near the C-terminal end of Prp2 eliminate its spliceosome binding and splicing activity. Here we show that a helicase-associated protein interacts with this domain and that this interaction contributes to the splicing process. First, a genome-wide yeast two-hybrid screen using Prp2 as bait identified Spp2, which contained a motif with glycine residues found in a number of RNA binding proteins. SPP2 was originally isolated as a genetic suppressor of a prp2 mutant. In a reciprocal screen, Spp2 specifically pulled out the C-terminal half of Prp2. Mutations in the Prp2 C-terminal 11-mer that disrupted function or spliceosome binding also disrupted Spp2 interaction. A screen of randomly mutagenized SPP2 clones identified an Spp2 protein with a mutation in the G patch that could restore interaction with Prp2 and enhanced splicing in a prp2 mutant strain. The study identifies a potential mechanism for Prp2 specificity mediated through a unique interaction with Spp2 and elucidates a role for a helicase-associated protein in the binding of a DEXD/H-box protein to the spliceosome.

The CLK/STY kinases are a family of dual-specificity protein kinases implicated in the regulation of cellular growth and differentiation. Some of the kinases in the family are shown to phosphorylate serine–arginine-rich splicing factors and to regulate pre-mRNA splicing. However, the actual cellular mechanism that regulates cell growth, differentiation, and development by CLK/STY remains unclear. Here we show that a functionally conserved CLK/STY kinase exists in Schizosaccharomyces pombe , and this orthologue, called Kic1, regulates the cell surface and septum formation as well as a late step in cytokinesis. The Kic1 protein is modified in vivo, likely by phosphorylation, suggesting that it can be involved in a control cascade. In addition, kic1 + together with dsk1 + , which encodes a related SR-specific protein kinase, constitutes a critical in vivo function for cell growth. The results provide the first in vivo evidence for the functional conservation of the CLK/STY family through evolution from fission yeast to mammals. Furthermore, since cell division and cell–cell interaction are fundamental for the differentiation and development of an organism, the novel cellular role of kic1 + revealed from this study offers a clue to the understanding of its counterparts in higher eukaryotes.

The unexpected low number of genes in the human genome has triggered increasing attention to alternative pre-mRNA splicing, and serine/arginine-rich (SR) proteins have been correlated with the complex alternative splicing that is a characteristic of metazoans. SR proteins interact with RNA and splicing protein factors, and they also undergo reversible phosphorylation, thereby regulating constitutive and alternative splicing in mammals and Drosophila. However, it is not clear whether the features of SR proteins and alternative splicing are present in simple and genetically tractable organisms, such as yeasts. In the present study, we show that the SR-like proteins Srp1 and Srp2, found in the fission yeast Schizosaccharomyces pombe, interact with each other and the interaction is modulated by protein phosphorylation. By using Srp1 as bait in a yeast two-hybrid analysis, we specifically isolated Srp2 from a random screen. This Srp interaction was confirmed by a glutathione-S-transferase pull-down assay. We also found that the Srp1—Srp2 complex was phosphorylated at a reduced efficiency by a fission yeast SR-specific kinase, Dis1-suppression kinase (Dsk1). Conversely, Dsk1-mediated phosphorylation inhibited the formation of the Srp complex. These findings offer the first example in fission yeast for interactions between SR-related proteins and the modulation of the interactions by specific protein phosphorylation, suggesting that a mammalian-like SR protein function may exist in fission yeast.

Introns are removed from nuclear messenger RNA precursors through two sequential phospho-transesterification reactions in a dynamic RNA–protein complex called the spliceosome1,2. But whether splicing is catalysed by small nuclear RNAs3,4 in the spliceosome is unresolved. As the spliceosome is a metalloenzyme5,6,7, it is important to determine whether snRNAs coordinate catalytic metals. Here we show that yeast U6 snRNA coordinates a metal ion that is required for the catalytic activity of the spliceosome. With Mg2+, U6 snRNA with a sulphur substitution for the pro-RP or pro-SP non-bridging phosphoryl oxygen of nucleotide U80 reconstitutes a fully assembled yet catalytically inactive spliceosome. Adding a thiophilic ion such as Mn2+ allows the first transesterification reaction to occur in the U6/sU80(SP)- but not the U6/sU80(RP)-reconstituted spliceosome. Mg2+ competitively inhibits the Mn2+-rescued reaction, indicating that the metal-binding site at U6/U80 exists in the wild-type spliceosome and that the site changes its metal requirement for activity in the SP spliceosome. Thus, U6 snRNA contributes to pre-messenger RNA splicing through metal-ion coordination, which is consistent with RNA catalysis by the spliceosome.

Arginine/serine-rich (RS) domain-containing proteins and their phosphorylation by specific protein kinases constitute control circuits to regulate pre-mRNA splicing and coordinate splicing with transcription in mammalian cells. We present here the finding that similar SR networks exist in Schizosaccharomyces pombe. We previously showed that Dsk1 protein, originally described as a mitotic regulator, displays high activity in phosphorylating S. pombe Prp2 protein (spU2AF59), a homologue of human U2AF65. We now demonstrate that Dsk1 also phosphorylates two recently identified fission yeast proteins with RS repeats, Srp1 and Srp2, in vitro. The phosphorylated proteins bear the same phosphoepitope found in mammalian SR proteins. Consistent with its substrate specificity, Dsk1 forms kinase-competent complexes with those proteins. Furthermore, dsk1(+) gene determines the phenotype of prp2(+) overexpression, providing in vivo evidence that Prp2 is a target for Dsk1. The dsk1-null mutant strain became severely sick with the additional deletion of a related kinase gene. Significantly, human SR protein-specific kinase 1 (SRPK1) complements the growth defect of the double-deletion mutant. In conjunction with the resemblance of dsk1(+) and SRPK1 in sequence homology, biochemical properties, and overexpression phenotypes, the complementation result indicates that SRPK1 is a functional homologue of Dsk1. Collectively, our studies illustrate the conserved SR networks in S. pombe consisting of RS domain-containing proteins and SR protein-specific kinases and thus establish the importance of the networks in eucaryotic organisms.

In this paper, TiSix as a new material suitable for using as an embedded layer for an attenuated phase shift mask (APSM) is presented. TiSix thin film was formed by plasma sputtering under Ar gas of 26 sccm. The related characteristics of TiSix at 365 nm (i-line) wavelength are as follows: n (refractive index) approximately 3.6; k (extinction coefficient) approximately 1.2; R percent (reflection) approximately 36, (rho) (resistivity 1.0 approximately 8.3 (mu) (Omega) -cm (depends on substrate). For required phase shift degree (theta) equals 180 degrees, calculated thickness d180 is 3.6 which is suitable for APSM. T percent under visible wavelength at thickness d180 is 14 approximately 17 which is suitable for mask's alignment by laser beam. TiSix film has good electrical conductivity, therefore, suitable for e-beam direct-write in patterning mask. TiSix film is also highly resistant to strong acid, suitable for fabrication of mask.© (1996) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Unlike autocatalyzed self-splicing of group I or group II introns, the removal of pre-mRNA introns in vitro occurs in the spliceosome. The spliceosome is a multicomponent complex composed of pre-mRNA, small nuclear ribonucleoprotein particles, and protein factors. ATP is required for the assembly of the spliceosome and both transesterification reactions. An RNA-dependent ATPase, the product of the yeast PRP2 gene, has been shown to be involved in the first transesterification of pre-mRNA splicing but not in spliceosome assembly. By using ATP analogs, we show that hydrolysis of ATP, mediated through a PRP2-dependent step, is required for the first catalytic event of pre-mRNA splicing. Furthermore, by using a two-step purification procedure, we have isolated a PRP2-containing spliceosome within which the first transesterification readily occurs after the addition of ATP. No additional macromolecules were required. Our results suggest that PRP2 binds to the spliceosome, interacting with an unidentified RNA species in the spliceosome, hydrolyzing ATP and allowing splicing to proceed. We postulate that PRP2 may function to induce a conformational change within the spliceosome. Alternatively, PRP2 may be involved in a proofreading step prior to splicing.

Synthetic actin and CYH2 pre-mRNAs containing a single intron are accurately spliced in a soluble whole cell extract of yeast. Splicing in vitro requires ATP. The excised intron is released as a lariat in which an RNA branch connects the 5' end of the molecule to the last A in the "intron conserved sequence" UACUAAC. Two other discrete RNA species produced during splicing in vitro may represent reaction intermediates: free, linear exon 1 and a form of the intron lariat extending beyond the 3' splice site to include exon 2. Both lariat forms correspond to molecules previously shown to be produced during yeast pre-mRNA splicing in vivo.

Transcripts from an ADC1-actin gene fusion containing the yeast actin intron are efficiently spliced in vivo. Four distinct forms of the excised actin intron are found in poly(A)- RNA from yeast carrying this transcription unit on a multicopy plasmid. Two of them migrate abnormally slowly in polyacrylamide gels, and one of these contains a block to reverse transcription at the conserved UACUAAC sequence (TACTAAC box). We also detect a larger RNA in the poly(A)+ fraction with abnormal gel mobility and the same reverse transcriptase block. Analysis of the major species of excised actin intron, labeled with 32P in vivo, reveals the presence of a branch at the third A in the UACUAAC sequence. This A is linked via a 3'-5' phosphodiester bond to the downstream C, and via a 2'-5' phosphodiester bond to a G, presumably from the 5' end of the intron. Thus this intron RNA is in the form of a lariat.

A novel family of large, imperfectly repeated DNA sequences has been found in Escherichia coli. Two members of this family, rhsA and rhsB, occur as direct repeats, flanking the pit glyS xyl segment of the chromosome. Unequal sister-chromatid crossing over between rhsA and rhsB accounts for the frequent tandem duplication of the glyS locus that has been observed by various workers. This unequal recombination is recA -dependent. The rhs A locus is operationally defined as the segment between xyl and mtl that is repeated at other chromosomal locations. Using this definition, rhsA extends minimally 5500 base-pairs; 3800 base-pairs of rhs A are sufficiently homologous to rhsB to form an S1 nuclease-resistant heteroduplex with it. The rhsA sequence also exhibits internal repetition. At least one additional rhs sequence occurs in the E. coli chromosome unlinked to either rhs A or rhsB. Southern analysis of restriction digests of genomic DNA from E. coli strains C and B/5 showed that both of these strains have rhs hybridizable patterns similar to strain K-12, but the rhs sequence is absent in Salmonella typhimurium. The function of the rhs sequences has not been discovered. In the course of this work we developed a technique, termed “transductional walking”, by which chromosomal DNA adjacent to a previously cloned DNA segment can be cloned through genetic procedures.

We report a correction to the Escherichia coli K-12 genetic map. The order of genes near minute 80 was found to be xyl-mtl-lct, not xyl-lct-mtl. The mapping utilized two different Tn10 insertion mutations, zia-138::Tn10 between xyl and mtl and zib-137::Tn10 distal to lct.

The yeast rna mutations ( rna 2– rna 11) are a set of temperature-sensitive mutations that result in the accumulation of intron-containing mRNA precursors at the restrictive temperature. We have used the yeast in vitro splicing system to investigate the role of products of the RNA genes in mRNA splicing. We have tested the heat lability of the in vitro mRNA splicing reaction in extracts isolated from mutant and wild-type cells. Extracts isolated from seven of the nine rna mutants demonstrated heat lability in this assay, while most wild-type extracts were stable under the conditions utilized. We have also demonstrated that heat inactivation usually results in the specific loss of an exchangeable component by showing that most combinations of heat-inactivated extracts from different mutants complement one another. In three cases ( rna 2, rna 5, and rna 11), the linkage of the in vitro defect to the rna mutations was ascertained by a combination of reversion, tetrad, and in vitro complementation analyses. Furthermore, each heat-inactivated extract was capable of complementation by at least one fraction of the wild-type splicing system. Thus many of the RNA genes are likely to code for products directly involved in and essential for mRNA splicing.

The cDNA for the two domain globin from the clam, Barbatia reeveana, has been cloned and largely sequenced. The results show that the amino acid sequences which correspond to the E and F helices in vertebrate globins are 100% identical in the two domains even though other parts of the domains differ substantially. This finding is consistent with the possibility that the E and F helices form important intersubunit contacts in the clam hemoglobin.

We have previously shown that extracts prepared from most of the yeast temperature-sensitive rna mutants are heat sensitive for pre-mRNA splicing in vitro, and that the products of the corresponding RNA genes are essential for the early stages of the splicing region. In this report, we demonstrate that most heat-inactivated mutant extracts do not form the spliceosome, suggesting that their gene products are likely to be involved in spliceosome formation. Heat-inactivated rna2 extracts, on the other hand, do form a splicing-dependent 40S complex containing uncleaved pre-mRNA exclusively. The pre-mRNA in the 40S complex can be converted to the splicing products in the presence of ATP and complementing extracts. These results demonstrate that: (1) the 40S complex formed in heat-inactivated rna2 extracts is a spliceosome (termed the rna2 delta spliceosome), (2) the spliceosome is a functional intermediate in the splicing pathway, and (3) the splicing process can be dissected into two steps, spliceosome formation and cleavage-ligation reactions. Additional results indicate that at least two extrinsic factors, as well as the RNA2 gene product, are required for complementation of the rna2 delta spliceosome. A three-step mechanism for nuclear pre-mRNA splicing in yeast is proposed.

Additional file 4: Figure S3. Alternative splicing of apoptosis genes.

Additional file 6: Figure S5. Alternative splicing of CDC25C in TF-1 cells treated with CIS, CPT, or TSA.

Additional file 6: Figure S5. Alternative splicing of CDC25C in TF-1 cells treated with CIS, CPT, or TSA.

Additional file 4: Figure S3. Alternative splicing of apoptosis genes.

Additional file 1. Supplementary information: supplemental figure legends, supplemental references, and Table S1.

Additional file 8: Figure S7. Cell cycle analysis of SRSF2 mutant cells and the effect of an apoptosis inhibitor on CDC25C alternative splicing.

Additional file 1. Supplementary information: supplemental figure legends, supplemental references, and Table S1.

Additional file 8: Figure S7. Cell cycle analysis of SRSF2 mutant cells and the effect of an apoptosis inhibitor on CDC25C alternative splicing.