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PubMed2005

Slowing down the Ras lane: miRNAs as tumor suppressors?

MicroRNAs (miRNAs) are small noncoding transcripts that regulate gene expression by promoting the degradation of transcribed messages or by inhibiting translation. Although bioinformatic approaches suggest that miRNAs may regulate the expression of a large fraction of the genome, the determination of miRNA gene targets and biological functions has been comparatively limited. Emerging studies suggest that many miRNAs may participate in human disease, including oncogenesis; but for the most part, the observations have been correlative. A recent study by Johnson and colleagues indicates that the let-7 miRNA negatively regulates the oncogenic family of Ras guanosine triphosphatases in both Caenorhabditis elegans and human tumor cell lines, suggesting that let-7 may act as a tumor suppressor. This work raises several important questions: Can other miRNAs act as tumor suppressors or oncogenes? Is miRNA deregulation a critical aspect of tumor development and maintenance? A number of recent studies have begun to address some of these functional questions, providing the field with a greater understanding of the role of miRNAs in cancer.

Animals↗

PubMed1983

Cloning the heavy chain of human HLA-DR antigen using synthetic oligodeoxyribonucleotides as hybridization probes.

The recent development of the amino acid microsequence technique allows us to obtain partial sequence information using an extremely small amount of protein. Two sets of mixed oligonucleotide probes were chemically synthesized using the amino acid sequence information for the heavy chain of human HLA-DR antigen obtained by the microsequence technique. These two hybridization probes were used to screen cDNA clones constructed from cytoplasmic poly(A)+ mRNA from a human B lymphoblastoid homozygous cell line (LG-2). Of the 10,000 clones screened, two clones hybridized with the probes. DNA sequence analysis showed that the longer one of the two cDNA clones was 1183 nucleotides long, including the entire coding region, the signal peptide region, and the complete 3'-noncoding region. The deduced amino acid sequence of the HLA-DR alpha chain is identical to that of other cell lines with a different HLA-DR typing. However, several nucleotide differences are found in the 3'-untranslated region compared with that of other DR haplotypes.

Amino Acid Sequence↗

PubMed2001

Imprinting defects in mouse embryos: stochastic errors or polymorphic phenotype?

Defects in expression of imprinted genes are believed to cause developmental abnormalities and play a role in carcinogenesis. To determine whether spontaneous imprinting defects may occur in mouse embryos, we studied the expression of two imprinted genes H19 and Igf2 in individual postimplantation 7.5 d.p.c. and 8.5 d.p.c. embryos. Biallelic expression of H19 was found in 1.6% of the embryos, whereas biallelic expression of Igf2 was found in 0.5% of the embryos. The loss of H19 imprinting (LOI) observed in a small fraction of early postimplantation embryos may be purely stochastic. Alternatively, since we never observed it in an inbred background, it may depend on genetic factors acting in trans. Either mechanism could explain the occurrence of polymorphic imprinting as well as the genesis of sporadic imprinting defects, including cancer. The frequency of LOI of H19 was higher than the incidence of sporadic imprinting disorders in humans (about 1 in 20,000). This contradiction may be explained by different incidence of imprinting errors in different imprinted regions of the genome, in different species, or by loss of the majority of nonmosaic embryos with imprinting defects before birth.

Alleles↗

PubMed1997

An alfalfa rubisco small subunit homologue shares cis-acting elements with the regulatory sequences of the RbcS-3A gene from pea.

A genomic clone of RbcS was isolated from an alfalfa (Medicago sativa L. cv. Apica) genomic library and characterized. Although this clone has structural features similar to a functional gene, the second exon is interrupted by a stop codon and thus is not fully translatable in the plant. Sequence analysis of the 5' and 3' noncoding regions of RbcSK-1A showed a high sequence homology to the flanking sequences of the RbcS-3A gene from pea. The regions of homology contain many important cis-regulatory elements shown to be essential for regulation of the RbcS-3A gene in pea. The promoter of this alfalfa rubisco clone was used in a translational fusion to test its ability to control the expression of the GUS reporter gene in an homologous nuclear background. High levels of GUS enzyme activity were recorded. These strong levels are comparable to some exceptionally high levels produced in other studies following the use of photosynthesis gene promoters in fusions with the GUS reporter gene.

Base Sequence↗

PubMed2004

Three-dimensional structure of C complex spliceosomes by electron microscopy.

The spliceosome is a multimegadalton RNA-protein machine that removes noncoding sequences from nascent pre-mRNAs. Recruitment of the spliceosome to splice sites and subsequent splicing require a series of dynamic interactions among the spliceosome's component U snRNPs and many additional protein factors. These dynamics present several challenges for structural analyses, including purification of stable complexes to compositional homogeneity and assessment of conformational heterogeneity. We have isolated spliceosomes arrested before the second chemical step of splicing (C complex) in which U2, U5 and U6 snRNAs are stably associated. Using electron microscopy, we obtained images of C complex spliceosomes under cryogenic conditions and determined a three-dimensional structure of a core complex to a resolution of 30 A. The structure reveals a particle of dimensions 27 x 22 x 24 nm with a relatively open arrangement of three primary domains.

Cryoelectron Microscopy↗

PubMed1988

Multiple species of myeloperoxidase messenger RNAs produced by alternative splicing and differential polyadenylation.

Three clones of full-length cDNA encoding human myeloperoxidase were isolated from a human leukemia HL-60 cell cDNA library in lambda gt10 and characterized. Analysis of the nucleotide sequence of one of the cDNA clones, lambda MP-H17, indicated that the cDNA contained 3207 bp with an open reading frame of 2238 bp, a 5' noncoding region of 159 bp, a 3' noncoding region of 800 bp, and a poly(A) tail of 10 bp. cDNA of the two other clones, lambda MP-H7 and lambda MP-H14, each contained insertions with shorter sequences of 96 and 82 bp, respectively, on the open reading frame of lambda MP-H17 cDNA. A myeloperoxidase genomic clone was isolated, and the structure of its 5' region was determined and compared with the structures of these cDNAs. The comparison revealed that the three cDNAs were derived from myeloperoxidase mRNAs produced by alternative splicing from a transcript of the single gene. Nucleotide sequence analysis of the 3' region of the cDNAs of several clones indicated that the mRNAs were polyadenylated at five different sites. Amino acid sequence determination of the amino-terminal and carboxy-terminal portions of the myeloperoxidase light and heavy chains revealed that, during processing of a precursor polypeptide into the mature protein, the amino-terminal polypeptide, the small peptide between the light and heavy chains, and the carboxy-terminal amino acid were excised.

Amino Acid Sequence↗

PubMed1992

Alternate use of divergent forms of an ancient exon in the fructose-1,6-bisphosphate aldolase gene of Drosophila melanogaster.

The fructose-1,6-bisphosphate aldolase gene of Drosophila melanogaster contains three divergent copies of an evolutionarily conserved 3' exon. Two mRNAs encoding aldolase contain three exons and differ only in the poly(A) site. The first exon is small and noncoding. The second encodes the first 332 amino acids, which form the catalytic domain, and is homologous to exons 2 through 8 of vertebrates. The third exon encodes the last 29 amino acids, thought to control substrate specificity, and is homologous to vertebrate exon 9. A third mRNA substitutes a different 3' exon (4a) for exon 3 and encodes a protein very similar to aldolase. A fourth mRNA begins at a different promoter and shares the second exon with the aldolase messages. However, two exons, 3a and 4a, together substitute for exon 3. Like exon 4a, exon 3a is homologous to terminal aldolase exons. The exon 3a-4a junction is such that exon 4a would be translated in a frame different from that which would produce a protein with similarity to aldolase. The putative proteins encoded by the third and fourth mRNAs are likely to be aldolases with altered substrate specificities, illustrating alternate use of duplicated and diverged exons as an evolutionary mechanism for adaptation of enzymatic activities.

Amino Acid Sequence↗

PubMed1997

Disrupted IGF2 promoter control by silencing of promoter P1 in human hepatocellular carcinoma.

Previous investigations have supported or indicated a stimulatory role of the insulin-like growth factor II gene (IGF2) in hepatocarcinogenesis. We have studied the transcript levels, promoter usage, and imprinting status of the ICF2 gene and its relationship to H19 in human hepatocellular carcinomas (HCCs) and liver tumor cell lines. The activity of the IGF2 promoter P1 was lost in about 70% of the cases (18 of 25). This is the most prominent abnormality regarding the IGF2 regulation in this study. Total IGF2 as well as promoter P3 transcription were up-regulated in a small group of the tumors. Twenty genetically informative cases were obtained from 26 cases, thus excluding the probability of loss of heterozygosity of the IGF2 gene. Among these, nine showed abnormal monoallelic expression of IGF2. One HCC and one HCC cell line proved loss of functional imprinting of IGF2. H19 and IGF2 were regulated in parallel, and expression levels were variable. Taken together, the disruption of the IGF2 promoter regulation, particularly the loss of P1 activity, is a common feature of human HCCs. The loss of P1 activity explains the frequent loss of biallelic IGF2 expression and may potentially be used as a diagnostic or monitoring marker for human HCC.

Alleles↗

PubMed2005

Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development.

MicroRNAs are small noncoding RNAs that control gene function posttranscriptionally through mRNA degradation or translational inhibition. Much has been learned about the processing and mechanism of action of microRNAs, but little is known about their biological function. Here, we demonstrate that injection of 2'O-methyl antisense oligoribonucleotides into early Drosophila embryos leads to specific and efficient depletion of microRNAs and thus permits systematic loss-of-function analysis in vivo. Twenty-five of the forty-six embryonically expressed microRNAs show readily discernible defects; pleiotropy is moderate and family members display similar yet distinct phenotypes. Processes under microRNA regulation include cellularization and patterning in the blastoderm, morphogenesis, and cell survival. The largest microRNA family in Drosophila (miR-2/6/11/13/308) is required for suppressing embryonic apoptosis; this is achieved by differential posttranscriptional repression of the proapoptotic factors hid, grim, reaper, and sickle. Our findings demonstrate that microRNAs act as specific and essential regulators in a wide range of developmental processes.

Animals↗

PubMed1998

Gene and cDNA structures of flounder insulin-like growth factor-I (IGF-I): multiple mRNA species encode a single short mature IGF-I.

To understand the comprehensive mechanisms of gene expression and processing for insulin-like growth factor-I (IGF-I) in vertebrates, we have investigated the gene organization, promoter and transcriptional initiation sites, alternative splicing and polyadenylating sites, and the cDNA structures of this gene in the Japanese flounder, Paralichthys olivaceus. The flounder IGF-I gene was found to be composed of five exons and four introns spanning 17.5 kb. By Northern blot analysis, two major mRNA classes of 4.7 kb and 2.9 kb were found in the liver. cDNA cloning and reverse transcription polymerase chain reaction (RT-PCR) analysis indicated that these two mRNA classes result from two different-sized 3'-noncoding regions generated by alternative usage of two polyadenylating signals. Further analysis by RT-PCR and sequencing revealed that these mRNA classes both contain two subclasses of mRNA encoding two forms of IGF-I prepropeptide, preproIGF-I-1 and preproIGF-I-2. The two forms of preproIGF-I share the identical signal peptide and mature IGF-I domain but contain different E domains as a result of alternative splicing in exon 3. The mature form of flounder IGF-I was found to comprise 68 amino acid residues, showing a small molecular weight, 7486. In the 5'-flanking region, one major and four minor transcription start sites have been identified by ribonuclease protection assay between -230 and -130 from the translation initiation codon, but no canonical TATA box or GC box was detected in their upstream regions up to -724. The results suggest that some unknown transcription initiation factors are functioning in the promotion of IGF-I gene expression.

Amino Acid Sequence↗

PubMed1998

First report on the systematic sequencing of the small genome of Encephalitozoon cuniculi (Protozoa, Microspora): gene organization of a 4.3 kbp region on chromosome I.

Belonging to a large group of parasitic amitochondrial protozoans (Microspora), Encephalitozoon cuniculi infects humans and other mammals. Because of its medical importance and small genome size (2.9 Mbp), we are systematically sequencing its smallest (217 kbp) chromosome. The shotgun cloning strategy now has produced the sequence of randomly dispersed contigs representing more than 180 kbp of this chromosome. The present report describes analysis of the 4.3 kbp contig, which includes the complete coding regions of dihydrofolate reductase (DHFR), thymidylate synthase (TS), and serine hydroxymethyl transferase (SHMT) genes and the partial coding region of an aminopeptidase (AP) gene. In contrast to the other reported protozoan genes, DHFR and TS are encoded by two different open reading frames (ORFs). The SHMT gene is the first one identified in a protozoan and corresponds to the cytosolic form of the enzyme. No introns were detected, and the intergenic noncoding regions do not exceed 50 bp. The mean GC content is close to 60%, and there is a G or C third-base codon bias. Transcription and translation initiation signals also are analyzed, and a model for the mRNA-ssu rRNA interactions is proposed.

Amino Acid Sequence↗

PubMed2002

Transport of Neuronal BC1 RNA in Mauthner Axons.

In neurons, localized RNAs have been identified in dendrites and axons; however, RNA transport in axons remains poorly understood. Here we analyzed axonal RNA transport in goldfish Mauthner neurons in vivo. BC1 RNA, a noncoding RNA polymerase III transcript that is targeted to dendrites in neurons of the rodent nervous system, was used as a probe for axonal RNA transport. Somata of Mauthner neurons were microinjected with various RNAs. Full-length BC1 RNA, but not control RNAs of similar length, was targeted to both axons and dendrites of Mauthner neurons. BC1 RNA was transported in the form of a rapidly advancing wave front that progressed along axons, in a microtubule-dependent manner, at a rate of 2 micrometer/sec. Whereas a BC1 5' segment of 65 nucleotides was transported to axons and dendrites in a way indistinguishable from full-length BC1 RNA, a BC1 3' segment of 60 nucleotides did not enter Mauthner cell processes to any significant extent. In the wake of the wave advancing through the axon, BC1 RNA was found localized to discrete, spatially delimited domains at the axonal surface. Such demarcated cortical concentrations of BC1 RNA could not be observed after disruption of F-actin organization in the axon. It is concluded that the specific delivery of BC1 RNA to spatially defined axonal target sites is a two-step process that requires the sequential participation of microtubules for long-range axial transport and of actin filaments for local radial transfer and focal accumulation in cortical domains.

5' Untranslated Regions↗

PubMed1995

The chicken beta A4- and beta B1-crystallin-encoding genes are tightly linked.

Analysis of the 5' flanking region of the chicken beta B1-crystallin-encoding gene (beta B1-cry) revealed regions of sequence homology with the bovine beta A4-crystallin-encoding gene (beta A4-cry). Subsequently, the chicken beta A4-cry cDNA sequence was determined, and it was demonstrated that beta A4- and beta B1-cry are linked head to head in the chicken chromosome with 2147 nucleotides (nt) of intergenic spacer. Chicken beta A4-cry contains six exons, with the first exon being noncoding. Chicken beta A4-cry is the smallest beta-cry ever described, due to the small size of its introns which range in length from 68 to 96 nt. While three polymorphisms were noted between some cDNA clones and the genomic sequence, Southern blot analysis demonstrated that beta A4-cry exists as a single copy in the chicken genome. Northern blot analysis indicated that beta A4-cry is a lens-specific transcript which is expressed at higher levels in the embryo than in the adult. The beta A4-cry mRNA is present at 400-fold lower levels than the beta B1-cry mRNA in the 14-day embryonic chicken lens, and at 2000-fold lower levels than the beta B1-cry mRNA in the adult lens. These results are consistent with the idea that the beta-cry family was once clustered in the chromosome as the gamma-cry family is today, and raises the possibility that the relatively low expression of beta A4-cry is mechanistically linked to the high expression of beta B1-cry in the chicken lens.

Animals↗

PubMed2005

Kaposi's sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells.

MicroRNAs (miRNAs) are an endogenously encoded class of small RNAs that have been proposed to function as key posttranscriptional regulators of gene expression in a range of eukaryotic species, including humans. The small size of miRNA precursors makes them potentially ideal for use by viruses as inhibitors of host cell defense pathways. Here, we demonstrate that the pathogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) encodes an array of 11 distinct miRNAs, all of which are expressed at readily detectable levels in latently KSHV infected cells. Individual KSHV miRNAs were expressed at up to 2,200 copies per cell. The KSHV miRNAs are expressed from what appears to be a single genetic locus that largely coincides with an approximately 4-kb noncoding sequence located between the KSHV v-cyclin and K12/Kaposin genes, both of which are also expressed in latently infected cells. Computer analysis of potential mRNA targets for these viral miRNAs identified a number of interesting candidate genes, including several mRNAs previously shown to be down-regulated in KSHV-infected cells. We hypothesize that these viral miRNAs play a critical role in the establishment and/or maintenance of KSHV latent infection in vivo and, hence, in KSHV-induced oncogenesis.

Base Sequence↗

PubMed2004

Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis.

MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are small noncoding RNAs that have recently emerged as important regulators of mRNA degradation, translational repression, and chromatin modification. In Arabidopsis thaliana, 43 miRNAs comprising 15 families have been reported thus far. In an attempt to identify novel and abiotic stress regulated miRNAs and siRNAs, we constructed a library of small RNAs from Arabidopsis seedlings exposed to dehydration, salinity, or cold stress or to the plant stress hormone abscisic acid. Sequencing of the library and subsequent analysis revealed 26 new miRNAs from 34 loci, forming 15 new families. Two of the new miRNAs from three loci are members of previously reported miR171 and miR319 families. Some of the miRNAs are preferentially expressed in specific tissues, and several are either upregulated or downregulated by abiotic stresses. Ten of the miRNAs are highly conserved in other plant species. Fifty-one potential targets with diverse function were predicted for the newly identified miRNAs based on sequence complementarity. In addition to miRNAs, we identified 102 other novel endogenous small RNAs in Arabidopsis. These findings suggest that a large number of miRNAs and other small regulatory RNAs are encoded by the Arabidopsis genome and that some of them may play important roles in plant responses to environmental stresses as well as in development and genome maintenance.

Animals↗

PubMed1991

Nucleotide sequence and evolution of coding and noncoding regions of a quail mitochondrial genome.

Segments of the Japanese quail mitochondrial genome encompassing many tRNA and protein genes, the small and part of the large rRNA genes, and the control region have been cloned and sequenced. Analysis of the relative position of these genes confirmed that the tRNA(Glu) and ND6 genes in galliform mitochondrial DNA are located immediately adjacent to the control region of the molecule instead of between the cytochrome b and ND5 genes as in other vertebrates. Japanese quail and chicken display another distinctive characteristic, that is, they both lack an equivalent to the light-strand replication origin found between the tRNA(Cys) and tRNA(Asn) genes in all vertebrate mitochondrial genomes sequenced thus far. Comparison of the protein-encoding genes revealed that a great proportion of the substitutions are silent and involve mainly transitions. This bias toward transitions also occurs in the tRNA and rRNA genes but is not observed in the control region where transversions account for many of the substitutions. Sequence alignment indicated that the two avian control regions evolve mainly through base substitutions but are also characterized by the occurrence of a 57-bp deletion/addition event at their 5' end. The overall sequence divergence between the two gallinaceous birds suggests that avian mitochondrial genomes evolve at a similar rate to other vertebrate mitochondrial DNAs.

Amino Acid Sequence↗

PubMed2005

Regulation of an intergenic transcript controls adjacent gene transcription in Saccharomyces cerevisiae.

Recent studies have revealed that transcription of noncoding, intergenic DNA is abundant among eukaryotes. However, the functions of this transcription are poorly understood. We have previously shown that in Saccharomyces cerevisiae, expression of an intergenic transcript, SRG1, represses the transcription of the adjacent gene, SER3, by transcription interference. We now show that SRG1 transcription is regulated by serine, thereby conferring regulation of SER3, a serine biosynthetic gene. This regulation requires Cha4, a serine-dependent activator that binds to the SRG1 promoter and is required for SRG1 induction in the presence of serine. Furthermore, two coactivator complexes, SAGA and Swi/Snf, are also directly required for activation of SRG1 and transcription interference of SER3. Taken together, our results elucidate a physiological role for intergenic transcription in the regulation of SER3. Moreover, our results demonstrate a mechanism by which intergenic transcription allows activators to act indirectly as repressors.

DNA, Intergenic↗

PubMed1994

Major human epididymis-specific gene product, HE3, is the first representative of a novel gene family.

Differential screening of a human epididymal cDNA library led to the isolation and characterization of a major epididymis-specific cDNA clone family, referred to as HE3. More detailed sequence and PCR analysis identified two different but homologous gene transcripts, HE3 alpha and HE3 beta. The former represents an mRNA of ca. 1 kb, encoding a putative small secretory polypeptide of 14903 MW. The HE3 beta transcript was only found as incomplete 3' fragments. Analysis of human genomic DNA by Southern blotting suggested the presence in the human genome of at least three independent HE3-related genes. Isolation of genomic clones for the HE3 alpha gene showed this to contain a single intron of 1.4 kb in the 5' noncoding region. Although genomic clones corresponding to HE3 beta could not be found, a third highly homologous gene, HE3 gamma, was identified as a potential pseudogene. Neither nucleotide nor encoded amino acid sequences of the HE3 gene family are related to any other known sequence in the central databases, and thus represents a novel human gene family, with at least three nonallelic members. Northern hybridization analysis showed that HE3 gene products are specifically expressed in the human epididymis, and not in any other tissue examined. Furthermore, except for the pig, no other nonprimate species has been identified to express homologous sequences in the epididymis. RNase protection assays showed that both the HE3 alpha and HE3 beta, but not the HE3 gamma genes, are expressed in the human epididymis.

Amino Acid Sequence↗