Protected RNA Fragments Research Articles

The splicing regulators RBM5 and RBM10 are subunits of the U2 snRNP engaged with intron branch sites on chromatin

Understanding the mechanisms of pre-mRNA splicing is limited by the technical challenges to examining spliceosomes invivo. Here, we report the isolation of RNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of mammalian cell nuclei. We found that these complexes contain U2 snRNP proteins and a portion of the U2 snRNA bound with protected RNA fragments that precisely map to intronic branch sites across the transcriptome. These U2 complexes also contained the splicing regulators RBM5 and RBM10. We found RBM5 and RBM10 bound to nearly all branch site complexes and not simply those at regulated exons. The deletion of a conserved RBM5/RBM10 peptide sequence, including a zinc finger motif, disrupted U2 interaction and rendered the proteins inactive for the repression of many alternative exons. We propose a model where RBM5 and RBM10 regulate splicing as components of the U2 snRNP complex following branch site base pairing.

Mammalian RNA Decay Pathways Are Highly Specialized and Widely Linked to Translation.

SummaryRNA decay is crucial for mRNA turnover and surveillance and misregulated in many diseases. This complex system is challenging to study, particularly in mammals, where it remains unclear whether decay pathways perform specialized versus redundant roles. Cytoplasmic pathways and links to translation are particularly enigmatic. By directly profiling decay factor targets and normal versus aberrant translation in mouse embryonic stem cells (mESCs), we uncovered extensive decay pathway specialization and crosstalk with translation. XRN1 (5′-3′) mediates cytoplasmic bulk mRNA turnover whereas SKIV2L (3′-5′) is universally recruited by ribosomes, tackling aberrant translation and sometimes modulating mRNA abundance. Further exploring translation surveillance revealed AVEN and FOCAD as SKIV2L interactors. AVEN prevents ribosome stalls at structured regions, which otherwise require SKIV2L for clearance. This pathway is crucial for histone translation, upstream open reading frame (uORF) regulation, and counteracting ribosome arrest on small ORFs. In summary, we uncovered key targets, components, and functions of mammalian RNA decay pathways and extensive coupling to translation.

Abstract GS2-01: Discovery and characterization of an estrogen bound LncRNA in late-Stage breast cancer

Abstract Breast cancer is the second most common newly diagnosed cancer and the second leading cause of cancer death among women in the United States. Despite the proven benefits of adjuvant endocrine therapy in women with hormone receptor positive breast cancer, relapses still occur over 5 years after initial treatment with endocrine therapy, referred to as late-stage relapse. Currently, the mechanisms of driving late-stage relapse are poorly characterized. To date, breast cancer research has primarily focused on protein-coding genes thereby missing the emerging class of long intergenic non-coding RNAs (lncRNAs) that may serve as critical regulators of relapse. Furthermore, the current understanding of lncRNA function is still in its infancy representing a critical gap for translating lncRNA discoveries into real-world applications to benefit patient care. Several well-described examples indicate that lncRNAs may be master epigenetic regulators in cancer biology through their interactions with proteins regulating target gene expression. Therefore, we hypothesize that lncRNAs may interact with estrogen receptor alpha 1 (ESR1) to regulate genes promoting late-stage relapse. To address this, we performed a transcriptome analysis of tumors from a unique cohort of 24 patients that had late-stage relapse to discover a novel set of lncRNAs, most of which have not yet been characterized. Next, we used RNA Immunoprecipitation coupled with transcriptome sequencing (RIP-Seq) to identify transcripts bound to ESR1 in T47D cells. We discovered 217 lncRNAs bound to ESR1 of which 50 were up-regulated in late-stage breast cancer. We chose to focus the most up-regulated lncRNA in late-stage relapse. Since it is an unannotated lncRNA we will refer to it as 'LAte-Stage relapse ESR1-Bound lncRNA 1', or LASER-1. To further understand the interplay between LASER-1 and ESR1, cells endogenously expressing LASER-1 were subjected to partial digestion to preserve lncRNA and protein interactions. Protected RNA fragments were subsequently immunoprecipitated with ESR1 and quantified by qPCR to reveal specific ESR1 interaction sites within LASER-1. Next, we observed increased expression of LASER-1 in ER+ breast cancer cell lines. Notably, LASER-1 expression was elevated in MCF7 long-term estrogen deprived (MCF7 LTED) cells -- that have amplified ESR1 -- relative to parental MCF7 cells. To demonstrate that LASER-1 promotes oncogenic phenotypes we transiently silenced LASER-1 in two cell lines with high endogenous expression of LASER-1 (including MCF LTED) and observed a decrease in cellular proliferation and invasion. Subsequent gene expression analysis after silencing LASER-1 altered mRNA and protein levels of critical cell cycle genes (i.e., p27). Overall, this is the first study to discover ESR1 bound lncRNAs that may be contributing to late-stage relapse in breast cancer. In the short-term, our ongoing research may lead to significant breakthroughs establishing the importance of LASER-1 as a master regulator in late-stage relapse. In the longer-term, we envision this research may lead to the development of novel therapeutics targeting LASER-1 with the potential for rapid clinical translation. Citation Format: Maher CA, Silva-Fisher J, Eteleeb A, Tang C, Perou C, Reis-Filho JS, Mardis ER, Ellis MJ. Discovery and characterization of an estrogen bound LncRNA in late-Stage breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS2-01.

Determinants of RNA Binding and Translational Repression by the Bicaudal-C Regulatory Protein

Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.

Detection of miRNA in Cell Cultures by Using Microchip Electrophoresis with a Fluorescence-Labeled Riboprobe

The analysis of a microRNA (miRNA), miR-222 isolated from the PC12 cell line, was performed by use of the ribonuclease (RNase) protection assay, cyanine 5 (Cy5)-labeled miR-222 riboprobe, and a Hitachi SV1210 microchip electrophoresis system, which can be used to evaluate the integrity of total RNA. The fluorescence intensity corresponding to the protected RNA fragment increased in a dose-dependent manner with respect to the complementary-strand RNA. More highly sensitive detection of miRNA by microchip electrophoresis than by conventional method using fluorescence-labeled riboprobe could be obtained in 180 s. An obvious increase in miR-222 expression induced by nerve growth factor in PC12 cells could be observed. These results clearly indicate the potential of microchip electrophoresis for the analysis of miRNA using RNase protection assay with a fluorescence-labeled riboprobe.

Ribonuclease protection assay on microchip electrophoresis

We describe the potential of microchip electrophoresis with a Hitachi SV1210, which can be used to evaluate the integrity of total RNA, for the analysis of mRNA expression. The ribonuclease (RNase) protection assay was performed by using microchip electrophoresis with cyanine 5 (Cy5) labeled 248-base antisense RNA probe (riboprobe) encoding adipose-type fatty acid binding protein (A-FABP) as the riboprobe. The fluorescence intensity corresponding to the protected RNA fragment increased in a dose-dependent manner with respect to the complementary strand RNA. Results were obtained in 120 s, and the same amount of Cy5-labeled antisense riboprobe as used in the conventional method can be used. Furthermore, 8 times more sensitive detection of mRNA by microchip electrophoresis could be obtained. An obvious increase in the mRNA expression of A-FABP, which is known as a differentiation marker of adipocytes, occurred during the adipocyte differentiation of 3T3-L1 cells. These results clearly indicate the potential of microchip electrophoresis for the analysis of mRNA expression in cells.

RNA channelling by the eukaryotic exosome

The eukaryotic exosome is a key nuclease for the degradation, processing and quality control of a wide variety of RNAs. Here, we report electron microscopic reconstructions and pseudo-atomic models of the ten-subunit Saccharomyces cerevisiae exosome in the unbound and RNA-bound states. In the RNA-bound structures, extra density that is visible at the entry and exit sites of the exosome channel indicates that a substrate-threading mechanism is used by the eukaryotic exosome. This channelling mechanism seems to be conserved in exosome-like complexes from all domains of life, and might have been present in the most recent common ancestor.

Association of RNA Helicase A with Human Immunodeficiency Virus Type 1 Particles

RNA helicase A (RHA) belongs to the DEAH family of proteins that are capable of unwinding double-stranded RNA structure. In addition to its involvement in the metabolism of cellular RNA, RHA has been shown to stimulate RNA transcription from the long terminal repeat promoter of human immunodeficiency virus type 1 (HIV-1) as well as to enhance Rev/Rev response element-mediated gene expression. In this study, we provide evidence that RHA associates with HIV-1 Gag in an RNA-dependent manner. This interaction results in specific incorporation of RHA into HIV-1 particles. Knockdown of endogenous RHA in virus producer cells leads to generation of HIV-1 particles that are less infectious in part as a result of restricted reverse transcription. Therefore, RHA represents the first example of cellular RNA helicases that participate in HIV-1 particle production and promote viral reverse transcription.

Distribution of Aromatase Cytochrome P450 Messenger Ribonucleic Acid in Adult Rhesus Monkey Brains1

The conversion of androgens to estrogens by aromatase cytochrome P450 (P450arom) is an important step in the mechanism of androgen action in the brain. However, the distribution of P450arom mRNA in adult rhesus monkey brains has not been studied because specific probes have not been available. To address this deficit, we cloned and sequenced a 455-basepair segment of the 5' coding region of the rhesus P450arom cDNA. Total RNA was extracted from a rhesus monkey placenta (Day 47 of gestation and subjected to reverse transcriptase (RT) polymerase chain reaction (PCR) using consensus oligonucleotide primers selected from published human and rat P450arom DNA sequences. The RT-PCR product was subcloned into a vector and sequenced. The monkey P450arom cDNA was 97% identical to the human sequence but shared only 86% homology with the rat sequence. We then developed a ribonuclease protection assay using a monkey P450arom cDNA and studied the distribution of P450arom mRNA in adult monkey brains. This assay protected two RNA fragments, one 455 nucleotides (nt) in length and the second approximately 300 nt. The relative distribution of P450arom mRNA (the 455-nt fragment) between brain areas of the adult (n = 3) was high in the bed nucleus of the stria terminalis > medial preoptic/anterior hypothalamus > amygdala; intermediate in the medial basal hypothalamus (infundibular nucleus, median eminence, ventromedial nucleus) > lateral preoptic/anterior hypothalamus; and low in the septum > lateral-dorsal-medial hypothalamus. P450arom mRNA was undetectable in cingulate and parietal cortex, hippocampus, and cerebellum. P450arom activity, as measured by the 3H2O assay, correlated well with the distribution of P450arom mRNA (the 455-nt protected fragment; r = 0.9) in the same tissues. A shorter protected RNA fragment was found in the medial basal hypothalamus, the bed nucleus of the stria terminalis, the amygdala, and the cingulate and parietal cortex but not in the other brain areas investigated. Its presence did not correlate with aromatase activity in brain tissue. This study describes the development of a ribonuclease protection assay using a monkey cDNA produced by RT-PCR and its usefulness for studying the distribution of P450arom mRNA in brains of nonhuman primates.

Detection and quantitation of specific mRNAs by ribonuclease protection assay using denaturing horizontal polyacrylamide gel electrophoresis: A radioactive and nonradioactive approach

A radioactive (32P) and nonradioactive (digoxigenin) ribonuclease protection assay (RPA) has been developed to detect mRNAs of housekeeping proteins and growth factors. A modification of polyacrylamide gel electrophoresis (PAGE) to simplify RPA is described. Both Cleangels (Pharmacia) and laboratory-cast polyacrylamide gels, in a denaturing, horizontal electrophoresis system, were used. The amount of toxic chemicals and waste was reduced, in comparison with sequencing gels normally used for RPA. The protected RNA fragments were shown to be well-separated, with sufficient sensitivity in this modified, quick gel system.

A sensitive method for detection of mutations--a PCR-based RNase protection assay.

Several techniques are currently available for detecting point mutations in DNA. The most widely used methods either use hazardous chemicals (chemical mismatch cleavage) or can detect mutations only in short (200- to 500-bp) fragments (single-stranded conformational polymorphism and denaturing gradient gel electrophoresis). In an effort to develop a sensitive and reliable method for the detection of mutations in large segments of DNA, a novel RNase protection assay using RNase I was developed. In this method, the desired portion of the gene is amplified by the polymerase chain reaction (PCR) using specific oligonucleotides and hybridized to a 32P-labeled RNA probe containing the wild-type sequence. The RNA/DNA hybrid is subsequently digested with RNase I, which cleaves the RNA at the mismatch sites. The protected RNA fragments are separated on a denaturing polyacrylamide-urea gel and detected by autoradiography. Four different RNA probes from two protein tyrosine phosphatases (PTP1C and PTP2C) were assayed using this procedure. Several mutants of the two enzymes were tested using wild-type RNA probes. Single-base changes involving all four bases at the mismatch site could be detected efficiently. The ability of this method to detect insertions and single-base deletions was also demonstrated. Using a PCR-based RNase protection assay, a single-base deletion in PTP1C in the motheaten mutation in mice could be detected. Using fragments amplified from genomic DNA, mice that were heterozygous for the motheaten mutation could be distinguished from wild type and homozygotes for this mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens regulate aromatase cytochrome P450 messenger ribonucleic acid in rat brain.

The conversion of androgens to estrogens by aromatase cytochrome P450 (P450AROM) is an important step in the mechanism of androgen action in the brain. In adult rats, P450AROM activity (AA) is regulated by androgens in the preoptic area and medial basal hypothalamus, but is constitutive in the amygdala. This study was undertaken to determine the distribution of P450AROM messenger RNA (mRNA) and AA in adult rat brain and examine the effects of steroid treatments on their concentrations in various brain regions. AA was determined by a sensitive assay that measures the production of 3H2O during the conversion of [1 beta-3H]androstenedione to estrone. P450AROM mRNA was measured by a ribonuclease protection assay using a RNA probe complementary to the 5'-coding region of rat P450AROM mRNA. The 32P-labeled P450AROM probe protected two mRNA fragments in brain tissues that expressed AA (preoptic area, medial basal hypothalamus, amygdala, and hippocampus). The larger protected RNA fragment was 430 nucleotides (nt) long and corresponded in size to the full-length protected complementary RNA, whereas the shorter protected RNA fragment was 300 nt long. Brain tissues that did not exhibit AA contained either the smaller protected RNA fragment (cingulate and parietal cortex) or no protected RNA (cerebellum). These results suggest that the 430-nt protected RNA fragment represents mRNA that encodes the functional P450AROM enzyme. In agreement with this conclusion, we found that immature rat ovaries that were stimulated with PMSG to synthesize estrogen contained only the 430-nt protected fragment. The levels of the 430-nt protected RNA fragment differed significantly between brain regions (amygdala > > preoptic area > medial basal hypothalamus > or = hippocampus) and were significantly correlated with AA (r = 0.994; P < 0.001). After castration, the concentrations of P450AROM mRNA and AA decreased significantly in the preoptic area and medial basal hypothalamus (P < 0.05), but not in the amygdala. Treatments with testosterone or dihydrotestosterone maintained P450AROM mRNA and AA at levels approximating those found in intact males. Although 17 beta-estradiol treatment increased AA in the preoptic area, it did not affect the P450AROM mRNA content. These results suggest that the increase in AA observed after exposure to androgens results from regulation of the transcription and/or stability of P450AROM mRNA. In contrast, estradiol appears to exert an effect on AA at the posttranscriptional level.(ABSTRACT TRUNCATED AT 400 WORDS)

Human osteosarcoma (U‐2 OS) cells express both insulin‐like growth factor‐I (IGF‐I) receptors and insulin‐like growth factor‐II/mannose‐6‐phosphate (IGF‐II/M6P) receptors and synthesize IGF‐II: Autocrine growth stimulation by IGF‐II via the IGF‐I receptor

Recently, insulin-like growth factor-I and -II (IGF-I and -II) have been implicated in the growth promotion of tumors in vivo and tumor cells in vitro. We have studied the human osteosarcoma cell line U-2 OS in order 1) to gain more insight into the growth promoting actions of the IGFs and 2) to establish an in vitro tissue culture model of IGF action in human tumor cells. Specific binding of 125I-IGF-I and 125I-IGF-II to IGF-I receptors and IGF-II/mannose-6-phosphate (M6P) receptors on U-2 OS cells was demonstrated in competitive binding experiments and in affinity crosslinking experiments. Western blotting of cell extracts confirmed the expression of the IGF-II/M6P receptor. In addition, in Northern blotting experiments using total RNA from U-2 OS cells IGF-I receptor RNA of 11 kb and IGF-II/M6P receptor RNA of approximately 9 kb were detected. Solution hybridization experiments confirmed the presence of IGF-I receptor and IGF-II/M6P receptor RNA. In a subset of experiments DNA synthesis was measured as 3H-thymidine uptake into cellular DNA of U-2 OS cells. Normal rat serum stimulated DNA synthesis maximally. IGF-I-deficient serum from hypophysectomized rats as well as IGF-I or IGF-II without serum were approximately twofold and tenfold, respectively, less potent than serum in stimulating 3H-thymidine uptake. The concentrations of IGF-I and IGF-II needed for half maximal stimulation of DNA synthesis corresponded well with the respective concentrations required for half maximal inhibition of 125I-IGF-I binding to U-2 OS cells. The anti-IGF-I receptor antibody alpha IR3 blocked the IGF-I and IGF-II stimulated increase of 3H-thymidine uptake. In addition, basal DNA synthesis was partially inhibited by the anti-IGF-I receptor antibody. These data suggest that U-2 OS cells synthesize and secrete IGF-like peptides. Northern blotting experiments confirmed that U-2 OS cells express an IGF-II RNA species of 5.3 kb but no IGF-I transcripts. In a series of RNase protection assays, protected RNA fragments were detected with an IGF-II riboprobe. Also, cell-conditioned medium from U-2 OS cells contained 1-2 ng/ml IGF-II immunoreactivity as measured in an IGF-binding protein blocked IGF-II radioimmunoassay. 1) U-2 OS cells express IGF-I and IGF-II/M6P receptors. 2) U-2 OS tumor cells respond to the addition of exogenous IGF-I and IGF-II with an increase of DNA synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Direct evidence for interaction of the conserved GTPase domain within 28 S RNA with mammalian ribosomal acidic phosphoproteins and L12.

A complex consisting of the acidic phosphoproteins P0, P1, and P2 (P proteins), L12, and RNA fragments was isolated from rat liver ribosomes after treatment with RNase T1 in the presence of EDTA. The complex was reactive with the anti-28 S RNA antibody specific for the highly conserved "GTPase domain" within 28 S rRNA. This suggests an association of these proteins with the RNA domain. To characterize this complex, the P proteins and L12 were isolated and tested for their binding specificity to the RNA by RNase T1 protection and gel retardation assays. Protein L12 and the P protein complex (P complex) both bound to rat 28 S rRNA and protected sequences comprising residues 1859-1921 and 1838-1936, respectively. The sequences overlap each other and lie in the GTPase domain. An in vitro transcript covering residues 1841-1936 of the 28 S rRNA as well as the protected RNA fragments also showed an ability to bind to the P complex and L12, and the binding was cooperative. RNA sequence elements within residues 1841-1936 required for protein binding were defined using site-directed mutagenesis. A unique internal loop including residues 1858 and 1859 and a distinct subregion comprising residues 1867-1914 in this domain were necessary for the binding of the P complex and L12, respectively. These results indicate that P proteins and L12 bind to restricted sites in the GTPase domain and that the complex constitutes the GTPase-related functional site in mammalian ribosomes.

Interactions between viral coat protein and a specific binding region on turnip crinkle virus RNA

The turnip crinkle virus coat protein binding sites in the ribonucleoprotein complex resulting from virion dissociation have been identified previously. In this study, RNA binding characteristics of viral coat protein to a region encompassing the protected RNA fragments Fa, Ff, and Fc (Fafc) have been investigated further using an RNA transcript (the Fafc fragment). These experiments have shown that coat protein requires no additional viral RNA elements to bind to this region. Such binding was shown to be specific for turnip crinkle virus coat protein using an ultra-violet light cross-linking assay. Gel mobility shift analyses demonstrated that the protein-RNA interactions produced two complexes: a homogeneous small ribonucleoprotein complex, and larger complexes which failed to migrate into gels. High salt and limiting protein concentrations favored the formation of the small ribonucleoprotein complex, whereas low salt and excess protein concentrations favored the larger complexes. RNA competition experiments demonstrated that small ribonucleoprotein complex formation coincided with specific RNA binding of the coat protein to the Fafc fragment. In addition, the coat protein possessed a poly(U)-binding site(s), which enabled it to interact with single-stranded RNA in a sequence non-specific manner to form large complexes. The results suggest that the coat protein contains both specific and non-specific RNA binding activities located at physically distinct sites. These results are consistent with the proposed assembly model for turnip crinkle virus.

Infection of human fetal dorsal root ganglion glial cells with human immunodeficiency virus type 1 involves an entry mechanism independent of the CD4 T4A epitope

Human immunodeficiency virus type 1 (HIV-1) has been implicated in the generation of acquired immunodeficiency syndrome-associated neurological dysfunction, and it is believed that the presence of CD4 in the nervous system may be involved in the susceptibility of selected neural cell populations to HIV-1 infection. We previously demonstrated (B. Wigdahl, R. A. Guyton, and P. S. Sarin, Virology 159:440-445, 1987) that glial cells derived from human fetal dorsal root ganglion (DRG) are susceptible to HIV-1 infection and subsequently express at least a fraction of the virus genome. In contrast to HIV-1 infection of CD4+ lymphocytes, which can be blocked by treatment with monoclonal antibodies directed against the HIV-1-binding region of CD4 (T4A epitope), treatment of human fetal DRG glial cells with similar antibodies resulted in only a slight reduction in HIV-1-specific gag antigen expression. In addition, preincubation of the HIV-1 inoculum prior to infection with HIV-1-neutralizing antiserum did not reduce HIV-1 gag antigen expression in these cells. Furthermore, we were unable to detect the synthesis or accumulation of the CD4 molecule in neural cell populations derived from DRG. However, a protected CD4-specific RNA fragment was detected in RNA isolated from human fetal DRG and spinal cord tissue by an RNase protection assay with a CD4-specific antisense RNA probe. RNA blot hybridization analysis of total cellular RNA isolated from human fetal DRG and spinal cord demonstrated specific hybridization to an RNA species that comigrated with the mature 3.0-kilobase CD4 mRNA as well as two unique CD4 RNA species with relative molecular sizes of approximately 5.3 and 6.7 kilobases. Furthermore, all three CD4-related RNA species were polyadenylated when isolated from human fetal spinal cord tissue. These data suggest that HIV-1 infection of human fetal DRG glial cells may proceed via a mechanism of viral entry independent of the T4A epitope of CD4.

Effect of ultraviolet light on the expression of genes for human U1 RNA.

Two types of UV-light-induced inhibitions of the synthesis of small nuclear RNA species U1, U2, U3, U4, and U5 were described previously: an immediate inhibition and a separate, delayed suppression that requires 1-2 hr of postirradiation cell incubation and UV doses that are about tenfold lower. In the present report, U1 RNA transcription in isolated nuclei from HeLa cells, assayed by RNAase T1 protection, reproduced the delayed inhibition. The sizes of the protected RNA fragments suggest that it is the initiation of U1 RNA transcription that is blocked during this inhibition. Transient expression of a marked human U1 RNA gene that contains 425 and 92 nucleotides of the 5' and 3' flanking sequences, respectively, showed delayed, but not immediate inhibition (while the endogenous U1 RNA genes exhibited immediate suppression). This indicates that continuity of the U1 gene flanking sequences beyond those segments and/or chromosomal integration of the U1 gene are not needed for the delayed inhibition, but may be required for the immediate inhibition. Irradiation of a U1 RNA gene, followed by its injection into Xenopus laevis oocyte nuclei, did not reproduce the immediate or delayed inhibitions. This suggests that direct UV radiation damage to DNA in the U1 RNA gene region is not the critical lesion in either the immediate or delayed UV-light-induced inhibitions of U1 RNA synthesis. In addition, the RNAase T1 protection pattern of transcripts synthesized in isolated nuclei from nonirradiated HeLa cells suggests that these cells may produce small amounts of U1 RNA molecules with variant nucleotide sequences in the mature region of the transcript.

Structural organization of ribonucleoproteins containing small nuclear RNAs from HeLa cells. Proteins interact closely with a similar structural domain of U1, U2, U4 and U5 small nuclear RNAs.

Abstract U 1 snRNP † isolated from HeLa cells and purified by centrifugation in cesium chloride contains a set of proteins that may be resolved into four/five polypeptides by gel electrophoresis. When this particle was submitted to extensive digestion with micrococcal nuclease, RNA fragments of about 25 nucleotides in length were obtained. Sequence analyses showed that these highly protected fragments were derived from the same region of the U 1 molecule, spanning positions 119 to 143. At low concentrations of nuclease, a longer fragment, from nucleotide 119 to the 3′ OH end, was also detected. U 1 core-resistant snRNP, isolated by high performance liquid chromatography, still contains all the protein components of the intact particle. When a less drastically purified U 1 snRNP containing, beside the four/five polypeptides remaining after centrifugation in cesium chloride, a set of at least three polypeptides of larger size, was digested with the nuclease, no other protected RNA fragment was detected. When a mixture of U 1 , U 2 , U 4 , U 5 and U 6 snRNPs, which contains the same four/five polypeptides as U 1 snRNP, was treated with micrococcal nuclease, protected fragments of snRNAs U 2 , U 4 and U 5 were found in addition to those derived from U 1 . No fragment derived from U 6 was found. In all cases, the region of snRNA shielded from nuclease attack corresponds to a distinctive feature of the molecule. It is a single-stranded region, comprising the sequence A(U) n G with n ≥ 3, bordered by two double-stranded stems. One of these stems includes the 3′ terminus of the RNA, except in the case of U 2 , where there are two stems instead of one on the 3′ side of the single-stranded stretch. Although a comparable structural domain exists also in U 6 snRNA, it does not contain the sequence A(U) n G which correlates well with the fact that no U 6 snRNA fragment seems to resist micrococcal nuclease digestion.

Binding to selected regions of reovirus mRNAs by a nonstructural reovirus protein.

When assembled into 13--19S particles, the reovirus nonstructural protein sigma-NS selectively binds single-stranded RNAs. Sedimentation analyses combined with binding to nitrocellulose membrane filters showed that 1--2 pmol of reovirus mRNAs from the large, medium, or small size classes saturated in vitro the binding site(s) on 13--19S particles containing 100 pmol of sigma-NS. All mRNA segments in each size class bound to particles, and no mRNAs in one size class excluded the binding of mRNAs in any other class. In competition experiments, the maximal binding of all reovirus mRNAs to particles of sigma-NS was achieved when medium and small mRNAs were bound before the large mRNAs. This preferred order of addition of mRNAs to sigma-NS resulted in a marked increase in the size of some of the complexes. This finding suggests that the addition of large mRNAs last to particles promoted the formation of complexes with more than one RNA segment bound per particle. The 13--19S particles of sigma-NS protected 20- to 40-nucleotide RNA fragments from nuclease digestion. At least one of the protected fragments from mRNAs of each size class included the 3' terminus; the remaining were from internal regions of the mRNAs. The protected RNA fragments rebound to particles during a second or third cycle of binding in a configuration in which they were fully protected from nuclease digestion. We conclude that binding of particles of sigma-NS to reovirus mRNAs was not at random sites but was to specific regions unique for members of each size class.

Polar Assembly of Clover Yellow Mosaic Virus

SUMMARY Clover yellow mosaic virus (CYMV) protein encapsidates its RNA at pH 7.5, low ionic strength and at a temperature of 25 °C. Protected RNA fragments extracted from the initiation complexes contained the cap structure m7GpppGp in the stoichiometric proportions expected for fragment sizes. These results were consistent with the location of the initiation site for assembly at or near the 5′ end of RNA; consequently, the maturation process is polar (5′ to 3′ direction). This strengthens the case for polar initiation as an alternative to internal initiation in the assembly of helical plant viruses.