The Role of miRNAs as Key Regulators in the Neoplastic Microenvironment.

The neoplastic microenvironment has been recognized to play a critical role in the development of cancer. Although a large body of evidence has established the importance of the cancer microenvironment, the manners of crosstalk between it and the cancer cells still remains unclear. Emerging mechanisms of communication include microRNAs (miRNAs). miRNAs are small noncoding RNA molecules that are involved in the posttranscriptional regulation of mRNA. Both intracellular and circulating miRNAs are differentially expressed in cancer and some of these alterations have been correlated with clinical patient outcomes. The role of miRNAs in the tumor microenvironment has only recently become a focus of research, however. In this paper, we discuss the influence of miRNAs on the tumor microenvironment as it relates to cancer progression. We conclude that miRNAs are a critical component in understanding invasion and metastasis of cancer cells.

Limitations of animal models of Parkinson's disease.

Most cases of Parkinson's disease (PD) are sporadic. When choosing an animal model for idiopathic PD, one must consider the extent of similarity or divergence between the physiology, anatomy, behavior, and regulation of gene expression between humans and the animal. Rodents and nonhuman primates are used most frequently in PD research because when a Parkinsonian state is induced, they mimic many aspects of idiopathic PD. These models have been useful in our understanding of the etiology of the disease and provide a means for testing new treatments. However, the current animal models often fall short in replicating the true pathophysiology occurring in idiopathic PD, and thus results from animal models often do not translate to the clinic. In this paper we will explain the limitations of animal models of PD and why their use is inappropriate for the study of some aspects of PD.

Conserved Wat1/Pop3 WD-repeat protein of fission yeast secures genome stability through microtubule integrity and may be involved in mRNA maturation.

Accurate chromosome segregation is dependent upon the integrity of mitotic spindles, which pull each pair of sister chromatids towards opposite poles. In this study, we have characterised fission yeast pop3-5235, a diploidising mutant that is impaired in genome stability. Pop3 is the same as Wat1, a conserved protein containing 7 WD repeats. Pop3/Wat1 has also been isolated from a two-hybrid screen as a binding partner to Prp2, the large subunit of the essential splicing factor U2AF. In wat1 mutants, the cellular amount of alpha-tubulin is decreased to very low levels, which results in compromised microtubules and spindles, consequently leading to unequal chromosome separation. Further analysis shows that, in spite of the binding between Wat1 and Prp2, Wat1 may not be involved directly in splicing reactions per se. Instead, we find that Wat1 is required for the maintenance of alpha-tubulin mRNA levels; moreover, transcript levels of genes other than the alpha-tubulin gene are also equally decreased in this mutant. Wild-type Wat1, but not the mutant protein, forms a large complex in the cell with several other proteins, suggesting that Wat1 functions as a structural linker in the complex. The results suggest that Wat1 plays a role in mRNA maturation as a coupling protein between splicing and synthesis and/or stabilisation.

Analysis of the splicing machinery in fission yeast: a comparison with budding yeast and mammals.

Based on genetic and bioinformatic analysis, 80 proteins from the newly sequenced Schizosaccharomyces pombe genome appear to be splicing factors. The fission yeast splicing factors were compared to those of Homo sapiens and Saccharomyces cerevisiae in order to determine the extent of conservation or divergence that has occurred over the billion years of evolution that separate these organisms. Our results indicate that many of the factors present in all three organisms have been well conserved throughout evolution. It is calculated that 38% of the fission yeast splicing factors are more similar to the human proteins than to the budding yeast proteins (>10% more similar or similar over a greater region). Many of the factors in this category are required for recognition of the 3' splice site. Ten fission yeast splicing factors, including putative regulatory factors, have human homologs, but no apparent budding yeast homologs based on sequence data alone. Many of the budding yeast factors that are absent in fission yeast are associated with the U1 and U4/U6.U5 snRNP. Collectively the data presented in this survey indicate that of the two yeasts, S.POMBE: contains a splicing machinery more closely reflecting the archetype of a spliceosome.

Mutations in the large subunit of U2AF disrupt pre-mRNA splicing, cell cycle progression and nuclear structure.

The prp2 gene of fission yeast has previously been shown to encode the large subunit of the splicing factor spU2AF. SpU2AF(59) is an evolutionarily conserved protein that has an arginine/serine-rich region and three RNA recognition motifs (RRMs). We have sequenced three temperature-sensitive alleles of prp2 and determined that the mutations result in single amino acid changes within one of the RRMs or between RRMs. All mutant alleles of prp2 have pre-mRNA splicing defects at the non-permissive temperature. Although the mutant strains are growth-arrested at 37 degrees C, they do not elongate like typical fission yeast cell cycle mutants. The DNA of the prp2(-) strains stains more intensely than a wild-type strain, suggesting that the chromatin may be condensed. Ultrastructural studies show differences in the mutant nuclei including a prominent distinction between the chromatin- and non-chromatin-enriched regions compared to the more homogenous wild-type nucleus. Two-hybrid assays indicate that some of the wild-type protein interactions are altered in the mutant strains. These results suggest that normal functioning of spU2AF(59) may be essential not only for pre-mRNA splicing but also for the maintenance of proper nuclear structure and normal cell cycle progression.

Cell-division-cycle defects associated with fission yeast pre-mRNA splicing mutants.

We have isolated six new pre-mRNA splicing mutants (prp) from a collection of temperature-sensitive (ts-) Schizosaccharomyces pombe strains. The prp mutants are defective in the splicing of both messenger RNA and U6 small nuclear RNA precursors. A single recessive mutation is responsible for both the ts- growth and the splicing phenotypes in each of the prp mutants. The six prp mutations are unlinked and fall into separate complementation groups. Two are allelic with the previously described prp3 and prp4 mutations; the remaining four define the new alleles prp5-1, prp6-1, prp7-1, and prp9-1. The six mutants exhibit three splicing phenotypes: accumulation of unspliced precursor at the restrictive but not at the permissive temperature; accumulation of unspliced precursor at both the permissive and restrictive temperatures; and accumulation of unspliced precursor, the intron-exon lariat intermediate, and the intron lariat final product. In addition to their aberrant splicing phenotypes, the prp5-1 and prp6-1 mutants express classical cell-division-cycle defects, while prp7-1 exhibits an unusual hyphal morphology. These results suggest a connection between pre-mRNA splicing and the control of cell division in fission yeast.

A potential role for U2AF-SAP 155 interactions in recruiting U2 snRNP to the branch site.

Base pairing between U2 snRNA and the branchpoint sequence (BPS) is essential for pre-mRNA splicing. Because the metazoan BPS is short and highly degenerate, this interaction alone is insufficient for specific binding of U2 snRNP. The splicing factor U2AF binds to the pyrimidine tract at the 3' splice site in the earliest spliceosomal complex, E, and is essential for U2 snRNP binding in the spliceosomal complex A. We show that the U2 snRNP protein SAP 155 UV cross-links to pre-mRNA on both sides of the BPS in the A complex. SAP 155's downstream cross-linking site is immediately adjacent to the U2AF binding site, and the two proteins interact directly in protein-protein interaction assays. Using UV cross-linking, together with functional analyses of pre-mRNAs containing duplicated BPSs, we show a direct correlation between BPS selection and UV cross-linking of SAP 155 on both sides of the BPS. Together, our data are consistent with a model in which U2AF binds to the pyrimidine tract in the E complex and then interacts with SAP 155 to recruit U2 snRNP to the BPS.

Molecular characterization of a novel fission yeast gene spUAP2 that interacts with the splicing factor spU2AF59.

A protein essential for pre-mRNA splicing, the U2 auxiliary factor (U2AF), is composed of a large and small subunit. Previously we cloned and characterized both subunits, spU2AF59 and spU2AF23, from fission yeast. We now report a novel U2AF-associated-protein, spUAP2, which interacts with both subunits. SpUAP2 contains a classical and a degenerate RNA recognition motif (RRM), both of which are required for interaction with spU2AF59. Interaction also requires the arginine/serine-rich region and the first RRM of spU2AF59. A null allele of the gene for spUAP2 is lethal.

BTF3 is evolutionarily conserved in fission yeast.

BTF3 is a protein initially identified in HeLa cells that may be involved in the initiation of transcription. Although its specific role in transcription is unclear, BTF3 can form a stable complex with RNA polymerase II. Recently, BTF3 has also been shown to bind to nascent polypeptide chains. We have cloned a homolog of BTF3 from the fission yeast, Schizosaccharomyces pombe. This homolog, spBTF3, encodes a putative 151 amino acid protein that shares 72% similarity with human BTF3, 73% similarity with the Caenorhabditis elegans homolog and between 52 and 53% similarity with the Saccharomyces cerevisiae homologs, EGD1 and BTT1.

The small subunit of the splicing factor U2AF is conserved in fission yeast.

The human splicing factor U2 auxiliary factor (hsU2AF) is comprised of two interacting subunits of 65 and 35 kDa. Previously we identified the Schizosaccharomyces pombe homolog, spU2AF59, of the human large subunit. We have screened a fission yeast cDNA library in search of proteins that interact with spU2AF59 using the yeast two-hybrid system and have identified a homolog of the hsU2AF35 subunit. The S. pombe U2AF small subunit is a single copy gene that encodes a protein which shares 55% amino acid identity and 17% similarity with the human small subunit. Unlike the human protein, the yeast protein lacks an arginine/serine-rich region. The predicted molecular mass of the spU2AF small subunit is 23 kDa. The region of spU2AF59 that interacts with spU2AF23 is similar to the region in which the human small and large subunits interact.

The evolutionary conservation of the splicing apparatus between fission yeast and man.

The removal of intervening sequences from pre-mRNA is an important step in gene regulation. Pre-mRNA processing takes place within the spliceosome, a dynamic structure composed of small nuclear RNA (snRNA) and proteins. The function of the spliceosome is currently being studied in many eukaryotic systems including mammal and yeast. Here we review pre-mRNA splicing in fission yeast and man and propose that spliceosomal structure and function has been evolutionarily conserved between the two organisms.

U2AF homolog required for splicing in vivo.

Several fission yeast temperature-sensitive mutants defective in pre-mRNA processing (prp- mutants) at the nonpermissive temperature have been identified. Here, the prp2+ gene has been cloned by its ability to complement the temperature-sensitive growth defect of a prp2- mutant. The gene also corrects the pre-mRNA splicing defect of prp2- mutants and encodes a 59-kilodalton polypeptide (PRP2). A molecular characterization indicates that PRP2 is a previously uncharacterized yeast splicing factor with extensive similarity to the mammalian splicing factor U2AF65. Thus, this study provides evidence that a U2AF homolog participates in RNA processing in vivo.

Differential distribution of factors involved in pre-mRNA processing in the yeast cell nucleus.

The yeast cell nucleus has previously been shown to be divided into two regions by a variety of microscopic approaches. We used antibodies specific for the 2,2,7-trimethylguanosine cap structure of small nuclear ribonucleic acids (snRNAs) and for a protein component of small nuclear ribonucleoprotein particles to identify the distribution of small nuclear ribonucleoprotein particles within the yeast cell nucleus. These studies were performed with the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae. By using immunofluorescence microscopy and immunoelectron microscopy, most of the abundant snRNAs were localized to the portion of the nucleus which has heretofore been referred to as the nucleolus. This distribution of snRNAs is different from that found in mammalian cells and suggests that the nucleolar portion of the yeast nucleus contains functional domains in addition to those associated with RNA polymerase I activity.

Schizosaccharomyces U6 genes have a sequence within their introns that matches the B box consensus of tRNA internal promoters.

The gene for the U6 small nuclear RNA (snRNA) in the fission yeast Schizosaccharomyces pombe is interrupted by an intron whose structure is similar to those found in messenger RNA precursors (pre-mRNAs) (1). This is the only known example of a split snRNA gene from any organism--animal, plant, or yeast. To address the uniqueness of the S. pombe U6 gene, we have investigated the structures of the U6 genes from five Schizosaccharomyces strains and three other fungi. A fragment of the U6 coding sequence was amplified from the genomic DNA of each strain by the polymerase chain reaction (PCR). The sizes of the PCR products indicated that all of the fission yeast strains possess intron-containing U6 genes; whereas, the U6 genes from the other fungi appeared to be uninterrupted. The sequences of the Schizosaccharomyces U6 gene fragments revealed that each had an intron of approximately 50 base pairs in precisely the same position. In addition to the splice sites and putative branch point regions, a sequence immediately upstream of the branch point consensus was found to be conserved in all of the Schizosaccharomyces U6 genes. This sequence matches the consensus for the B box of eukaryotic tRNA promoters. These results raise the interesting possibility that synthesis of U6 RNA in fission yeast might involve the use of internal promoter elements similar to those found in other genes transcribed by RNA polymerase III.

A mutation in a single gene of Schizosaccharomyces pombe affects the expression of several snRNAs and causes defects in RNA processing.

A bank of temperature sensitive (ts-) mutants of Schizosaccharomyces pombe was screened for snRNA expression mutants using an oligodeoxynucleotide that recognizes U2 RNA. One mutant with a novel phenotype was identified that has reduced steady-state levels of the spliceosomal snRNAs U1, U2, U4, U5 and U6. In addition, the mutant exhibits a temperature-dependent accumulation of aberrant U2 and U4 transcripts elongated at their 3' end. The steady-state concentration of the RNA component of RNase P is also reduced in the mutant, whereas the amount of U3 RNA, 7SL RNA, tRNA, rRNA and mRNA are the same as wild-type. Pre-mRNA, pre-tRNA and U6 RNA precursor processing are impaired in the mutant. Genetic analysis demonstrates that the snRNA defects are tightly linked to the ts- growth defect and are recessive. We have named this mutant snm1 to indicate a defect in snRNA maintenance. The data on snm1 suggest that a single trans-acting factor is essential for the maintenance of steady-state levels of several snRNAs and for proper 3' end formation of U2 and U4 RNAs.

Splicing of the U6 RNA precursor is impaired in fission yeast pre-mRNA splicing mutants.

U6 RNA is a member of a class of small abundant stable nuclear RNAs that are essential for splicing. In all species examined so far, the U6 RNA is a RNA polymerase III transcript. The U6 gene of the fission yeast Schizosaccharomyces pombe is unusual in that it is interrupted by an intron whose structure is similar to those found in pre-mRNAs. As part of our previous analysis of three S. pombe temperature sensitive pre-mRNA splicing mutants we examined their spliceosomal snRNA content. In contrast to the other snRNAs, the amount of U6 RNA is reduced at the restrictive temperature in all three of the mutants compared to the wild type. To investigate the cause of this reduction we have analyzed the efficiency of splicing of the U6 RNA precursor (U6 pre-RNA) in the pre-mRNA splicing mutants. At the restrictive temperature the ratio of unspliced U6 precursor to mature RNA is elevated in the mutants compared to the wild type grown under identical conditions, indicating a defect in U6 pre-RNA splicing. In this regard, the U6 RNA precursor behaves similarly to pre-mRNAs. Unspliced U6 pre-RNA was also detected in wild type cells under certain growth conditions.

Pre-mRNA splicing mutants of Schizosaccharomyces pombe.

A collection of temperature sensitive (ts-) mutants was prepared by chemical mutagenesis of a wild type Schizosaccharomyces pombe strain. To screen the ts- mutants for pre-mRNA splicing defects, an oligodeoxynucleotide that recognizes one of the introns of the beta-tubulin pre-mRNA was used as a probe in a Northern blot assay to detect accumulation of intron sequences. This screening procedure identified three pre-mRNA splicing mutants from 100 ts- strains. The three mutants are defective in an early step of the pre-mRNA splicing reaction; none accumulate intermediates. The precursors that accumulate at 37 degrees C are polyadenylated. Analysis of the splicing of another pre-mRNA showed that the mutations are not specific for beta-tubulin. The total RNA pattern in the three splicing mutants appears to be normal. In addition, the amounts of the spliceosomal snRNAs are not drastically changed compared to the wild type and splicing of pre-tRNAs is not blocked. Genetic analyses demonstrate that all three splicing mutations are tightly linked to the ts- growth defects and are recessive. Crosses among the mutants place them in three complementation groups. The mutants have been named prp1, prp2 and prp3.

Multiple phosphorylated forms of the product of the fission yeast cell division cycle gene cdc2+.

The 34 kilodalton protein product (p34) of the cdc2+ cell cycle control gene of Schizosaccharomyces pombe was expressed in bacteria. Monoclonal antibodies raised against this protein are capable of immunoprecipitating p34cdc2 from yeast lysates. Immunoprecipitates of [35S]methionine- and [32P]orthophosphate-labeled p34cdc2 were analyzed by two-dimensional gel electrophoresis. The cdc2+ gene product is homogeneous in size but resolves into seven species of differing charge. At least four of these species are phosphorylated. Phosphoamino acid analysis reveals that phosphorylation occurs mainly on threonine residues. The pattern of p34 phosphorylation is unaltered at the nonpermissive temperature in strains carrying temperature sensitive alleles of weel-50 and ran1-114 or in a strain overproducing the ran1+ gene product.

Identification of p34 and p13, human homologs of the cell cycle regulators of fission yeast encoded by cdc2+ and suc1+.

cdc2+ and CDC28 play central roles in the cell division cycles of the widely divergent yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. The genes encode protein kinases that show 62% protein sequence identity and are capable of cross-complementation. Monoclonal antibodies were raised against p34cdc2, and a subset recognize p36cdc28. The cross-reacting antibodies detected a 34 kd homolog of the p34cdc2/p36CDC28, protein in HeLa cells. Human p34 was also recognized by an affinity-purified polyclonal anti-p34cdc2 serum. Peptide mapping of p34cdc2, p36CDC28, and human p34 revealed complete conservation of four tryptophan residues in the three proteins. p34 thus appears to be closely related to the two yeast proteins. In addition, a p34 immune complex showed protein kinase activity in vitro, and HeLa cell p34 interacts with p13, the human homolog of the suc1+ gene product of S. pombe.

Characterization of DNA sequences associated with residual nuclei of Saccharomyces cerevisiae.

We have used two different approaches to determine whether particular DNA sequences are specifically associated with high-salt-treated residual nuclei of Saccharomyces cerevisiae. First, libraries of yeast DNA in phage lambda were probed with nick-translated total nuclear or residual nuclear DNA from unsynchronized yeast cells. None of the plaques gave a significantly stronger or weaker signal with the residual nuclear probe than with the total nuclear probe. Second, DNA was purified from whole nuclei or residual nuclei which had been isolated from cells in G1, G1/S, early S, or nuclear division. This DNA was "dot-blotted" and then probed with specific yeast DNA sequences. Ribosomal DNA was 2- to 3-fold enriched in residual nuclei in late G1, G1/S, and early S, and 2 microns plasmid DNA sequences were 3- to 5-fold depleted during nuclear division and early G1. However, ARS1, TRP1, CEN6, and a telomere sequence were neither enriched nor depleted at any time during the cell cycle.