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A second case has been found of a nematode gene involved in developmental timing that encodes a short, non-coding RNA. Both RNAs are expressed at specific times and appear to repress target genes by interacting with their 3' untranslated regions. A coincidence? Or does this pathway attract small RNA regulators?
Cells respond to changes in environmental conditions via orchestrated modifications in gene expression. For example, in response to heat shock, cells execute a program of gene-specific transcriptional activation and repression. Although the activation of genes upon heat shock has been widely studied, the mechanism of mRNA transcriptional repression upon heat shock is unexplained. Here we show that during the heat shock response in mouse cells, a small noncoding RNA polymerase III transcript, B2 RNA, associates with RNA polymerase II and represses transcription of specific mRNA genes. These studies define a unique transcriptional regulatory mechanism involving an RNA regulator and reveal how mRNA transcription is repressed upon heat shock. Moreover, we identify a function for B2 RNA, which is transcribed from short interspersed elements that are abundant in the mouse genome and historically considered to be 'junk DNA.'
A sensitive and quantitative in vitro analysis method was established to detect the nascent RNAs stimulated by angiogenin using the nuclei isolated from human umbilical vein endothelial cells (HUVE). Angiogenin was mixed with nuclei in the reaction buffer, then transcription was initiated by adding the NTPs mixture. The RNA products were measured quantitatively by [alpha-(32)P]CTP incorporation with a liquid scintillation counter, either after removing free isotope by using spin column, or cutting the electrophoresis lane and counting after autoradiography. It was found that the optimum reacting temperature was 30 degrees, the most suitable reaction time was 30 min for this system, and the transcription enhancement activity of angiogenin was dose-dependent with the feasible concentration being 1 mg/L. Higher concentration of angiogenin degraded the RNA products in the system, suggesting that there is a mechanism to control the entry and accumulation of angiogenin in the target cells, which ensured angiogenin to play its role properly in the cells. Based on the evidence that angiogenin bound to DNA in nucleolus and enhanced RNA transcription, it was proposed that angiogenin might act as a trans-acting factor in nucleus to regulate RNA transcription, and inhibition of angiogenin-stimulated RNA transcription might be a promising target for screening anti-angiogenesis inhibitor.
Messenger RNA regulation is a critical mode of controlling gene expression. Regulation of mRNA stability and translation is linked to controls of poly(A) tail length. Poly(A) lengthening can stabilize and translationally activate mRNAs, whereas poly(A) removal can trigger degradation and translational repression. Germline granules (for example, polar granules in flies, P granules in worms) are ribonucleoprotein particles implicated in translational control. Here we report that the Caenorhabditis elegans gene gld-2, a regulator of mitosis/meiosis decision and other germline events, encodes the catalytic moiety of a cytoplasmic poly(A) polymerase (PAP) that is associated with P granules in early embryos. Importantly, the GLD-2 protein sequence has diverged substantially from that of conventional eukaryotic PAPs, and lacks a recognizable RRM (RNA recognition motif)-like domain. GLD-2 has little PAP activity on its own, but is stimulated in vitro by GLD-3. GLD-3 is also a developmental regulator, and belongs to the Bicaudal-C family of RNA binding proteins. We suggest that GLD-2 is the prototype for a class of regulatory cytoplasmic PAPs that are recruited to specific mRNAs by a binding partner, thereby targeting those mRNAs for polyadenylation and increased expression.
We have examined the ability of the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), to regulate RNA polymerase III gene expression in Drosophila. Using nuclear run-on assays, we detected a 3-5-fold increase in tRNA synthesis following treatment of Drosophila Schneider S2 cells with TPA, whereas transcription from the actin 5C, fos-, and jun-related antigen promoters was unaffected. This response is rapid and transient, peaking at about a 45-min exposure of the cells to TPA, and dissipating after 60 min. We have reproduced this stimulation in vitro. Extracts prepared from cells treated with TPA show an approximate 10-fold increase in specific transcription using a 5 S RNA and various tRNA gene templates. The nonspecific transcription by RNA polymerase III in these extracts, however, is essentially unchanged. Mixing the extracts derived from uninduced and induced cells suggests that the TPA stimulation observed may be due to the increase of a positive-acting factor. These results are the first to demonstrate that a phorbol ester can induce RNA polymerase III gene expression. The ability to reproduce this activation in vitro will now allow us to assess the role of the transcription components in this regulatory event, and the biochemical consequence of this signaling pathway.
Alternative RNA processing of human calcitonin/CGRP pre-mRNA is regulated by an intronic enhancer element. Previous studies have demonstrated that multiple sequence motifs within the enhancer and a number of trans-acting factors play critical roles in the regulation. Here, we report the identification of TIAR as a novel player in the regulation of human calcitonin/CGRP alternative RNA processing. TIAR binds to the U tract sequence motif downstream of a pseudo 5' splice site within the previously characterized intron enhancer element. Binding of TIAR promotes inclusion of the alternative 3'-terminal exon located more than 200 nucleotides upstream from the U tract. In cells that preferentially include this exon, overexpression of a mutant TIAR that lacks the RNA binding domains suppressed inclusion of this exon. In this report, we also demonstrate an unusual novel interaction between U6 snRNA and the pseudo 5' splice site, which was shown previously to bind U1 snRNA. Interestingly, TIAR binding to the U tract sequence depends on the interaction of not only U1 but also U6 snRNA with the pseudo 5' splice site. Conversely, TIAR binding promotes U6 snRNA binding to its target. The synergistic relationship between TIAR and U6 snRNA strongly suggests a novel role of U6 snRNP in regulated alternative RNA processing.
Transcription of large rRNA precursor and 5S RNA were examined during encystment of Acanthamoeba castellanii. Both transcription units are down regulated almost coordinately during this process, though 5S RNA transcription is not as completely shut down as rRNA transcription. The protein components necessary for transcription of 5S RNA and tRNA were determined, and fractions containing transcription factors comparable to TFIIIA, TFIIIB, and TFIIIC, as well as RNA polymerase III and a 3'-end processing activity, were identified. Regulation of 5S RNA transcription could be recapitulated in vitro, and the activities of the required components were compared. In contrast to regulation of precursor rRNA, there is no apparent change during encystment in the activity of the polymerase dedicated to 5S RNA expression. Similarly, the transcriptional and promoter-binding activities of TFIIIC are not altered in parallel with 5S RNA regulation. TFIIIB transcriptional activity is unaltered in encysting cells. In contrast, both the transcriptional and DNA-binding activities of TFIIIA are strongly reduced in nuclear extracts from transcriptionally inactive cells. These results were analyzed in terms of mechanisms for coordinate regulation of rRNA and 5S RNA expression.
Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 has previously been shown to bind mouse hepatitis virus (MHV) RNA at the 3' end of both plus and minus strands and modulate MHV RNA synthesis. However, a mouse erythroleukemia cell line, CB3, does not express hnRNP A1 but still supports MHV replication, suggesting that alternative proteins can replace hnRNP A1 in cellular functions and viral infection. In this study, we set out to identify these proteins. UV cross-linking experiments revealed that several CB3 cellular proteins similar in size to hnRNP A1 interacted with the MHV RNA. These proteins were purified by RNA affinity column with biotinylated negative-strand MHV leader RNA and identified by mass spectrometry to be hnRNP A2/B1, hnRNP A/B, and hnRNP A3, all of which belong to the type A/B hnRNPs. All of these proteins contain amino acid sequences with strong similarity to the RNA-binding domains of hnRNP A1. Some of these hnRNPs have previously been shown to replace hnRNP A1 in regulating RNA splicing. These proteins displayed MHV RNA-binding affinity and specificity similar to those of hnRNP A1. hnRNP A2/B1, which is predominantly localized to the nucleus and shuttles between the nucleus and the cytoplasm, was shown to relocalize to the cytoplasm in MHV-infected CB3 cells. Furthermore, overexpression of hnRNP A/B in cells enhanced MHV RNA synthesis. Our findings demonstrate that the functions of hnRNP A1 in MHV RNA synthesis can be replaced by other closely related hnRNPs, further supporting the roles of cellular proteins in MHV RNA synthesis.
A central player in the Arabidopsis floral transition is the floral repressor FLC, the MADS-box transcriptional regulator that inhibits the activity of genes required to switch the meristem from vegetative to floral development. One of the many pathways that regulate FLC expression is the autonomous promotion pathway composed of FCA, FY, FLD, FPA, FVE, LD, and FLK. Rather than a hierarchical set of activities the autonomous promotion pathway comprises sub-pathways of genes with different biochemical functions that all share FLC as a target. One sub-pathway involves FCA and FY, which interact to regulate RNA processing of FLC. Several of the identified components (FY, FVE, and FLD) are homologous to yeast and mammalian proteins with rather generic roles in gene regulation. So why do mutations in these genes specifically show a late-flowering phenotype in Arabidopsis? One reason, found during the analysis of fy alleles, is that the mutant alleles identified in flowering screens can be hypomorphic, they still have partial function. A broader role for the autonomous promotion pathway is supported by a microarray analysis which has identified genes mis-regulated in fca mutants, and whose expression is also altered in fy mutants.
The rne gene of Escherichia coli encodes a 118 kDa protein that has ribonuclease E (RNase E) activity and binds RNA. A functional rne gene product is essential for cell viability and for the processing and/or decay of a variety of RNA species, including 9 S RNA, mRNA and RNAI, the antisense RNA regulator of ColE1-type plasmid replication. By testing the ability of different segments of the Rne protein to catalyze RNA cleavage and to bind RNA, we found that the N-terminal half (residues 1 to 498) of Rne contains a catalytic function sufficient for site-specific cleavage of oligoribonucleotides and complex RNAs. The C-terminal half of the protein, which contains both an arginine-rich region (residues 597 to 684) that we show binds RNA and a segment that is essential for cell viability (residues 844 to 1061), had no detectable endoribonucleolytic activity. Our results, which map the catalytic domain of RNase E, indicate the existence of discrete functional domains within the multifaceted Rne protein.
Liver RNA- and protein-degradation rates were measured after the induction of acute inflammation in the rat. A preliminary study determined changes in hepatic RNA and protein content 12, 18 and 24 h after a turpentine oil injection. The RNA content in turpentine-treated rats compared with pair-fed animals increased significantly and sharply from 12 h (+ 11%) to 18 h (+ 32%) and slightly thereafter (+ 37% at 24 h). The liver protein content was significantly enhanced only at 24 h (+ 11%) in response to inflammation. RNA-degradation rates were determined in livers perfused cyclically in situ for 15 min by measuring the accumulation of radioactive cytidine in the medium 60 h after in vivo labelling of RNA by [5-3H]cytidine instead of [6-14C]orotic acid, the most commonly used radioactive marker. Several validation procedures showed that the method employed was a valid alternative to the use of radioactive orotic acid. RNA-degradation rates, which mainly reflect rRNA breakdown, were significantly lower in the turpentine-treated rats than in respective pair-fed animals at 18 and 24 h (57 and 45% decrease respectively). Proteolysis rates measured at 24 h together with RNA breakdown by valine accumulation in the perfusion medium were not modified after turpentine treatment. The main factors known to regulate RNA degradation (amino acids, insulin/glucagon ratio) were measured in the portal blood 24 h after induction of acute inflammation. Of the known regulatory amino acids, only glutamine and to a lesser extent methionine were increased in the turpentine-treated rats as compared with their pair-fed counterparts. The insulin/glucagon molar ratio was similar in both groups. In conclusion, the reduced breakdown of RNA, especially rRNA, is largely responsible for the accumulation of hepatic RNA during acute inflammation. This inhibition of RNA degradation could possibly be related to the increase in glutamine.
Among the goals of RNA structural and functional genomics is determining structures and establishing the functions of a rich repertoire of simple sequence repeats in transcripts. These repeats are present in transcripts from their 'birth' in the nucleus to their 'death' in cytoplasm and have the potential of being involved in many steps of RNA regulation. The knowledge of their structural features and functional roles will also shed more light on the postulated mechanisms of RNA pathogenesis in a growing list of neurological diseases caused by simple sequence repeat expansions. Here, we discuss several different lines of research to support the hypothesis that the mechanism of RNA pathogenesis may be a more common phenomenon triggered or modulated also by abundant long normal repeats. We propose structures of the repeat regions in transcripts of genes involved in Triplet Repeat Expansion Diseases. We have classified the polymorphic repeat alleles of these genes according to their ability to form hairpin structures in transcripts, and describe the distribution of different structural forms of the repeats in the human population. We have also reported the results of a systematic survey of the human transcriptome to identify mRNAs containing triplet repeats and to classify them according to structural and functional criteria. Based on this knowledge, we discuss the putative wider role of triplet repeat RNA hairpins in human diseases. A hypothetical model is proposed in which long normal RNA hairpins formed by the repeats may also be involved in pathogenesis.
The removal of introns from precursor messenger RNAs occurs in a large complex, the spliceosome, that contains many proteins and five small nuclear RNAs (snRNAs). The snRNAs interact with the intron-containing substrate RNA and with each other to form a dynamic network of RNA interactions that define the intron and promote splicing. There is evidence that protein splicing factors play important roles in regulating RNA interactions in the spliceosome. PRP8 is a highly conserved protein that is associated in particles with the U5 snRNA and directly binds the substrate RNA in spliceosomes. UV crosslinking has been used to map the binding sites, and shows extensive interaction between PRP8 protein and the 5' exon prior to the first step of splicing and with the 3' splice site region subsequently. It is proposed that PRP8 protein may stabilize fragile interactions between the U5 snRNA and exon sequences at the splice sites, to anchor and align them in the catalytic centre of the spliceosome.
When starved for leucine, strains of Bacillus subtilis do not complete chromosome replication to the terminus. The amount of deoxyribonucleic acid (DNA) made poststarvation is characteristic of the strain. In this study, four strains differing in their DNA response were examined for ribonucleic acid (RNA) regulation during leucine starvation. Each of the strains was judged to be stringent for RNA control based on the amount of RNA made poststarvation. Sucrose gradient profiles on RNA made with and without leucine starvation support this conclusion. Accumulation of guanosine tetraphosphate during leucine starvation showed no correlation with the amount of DNA synthesized. We concluded that modulation control of DNA synthesis during leucine starvation is independent of RNA control.
Recently, we identified a specific DNA sequence from the gastrin gene that regulates RNA polymerase II transcription termination in vivo. In the studies presented here, we examined the processing and termination activity of this sequence in vitro. When present in an in vitro synthesized RNA, this sequence (U9A2U5AU4AU4AU5) does not serve as an RNA processing signal on incubation with HeLa whole-cell extract. However, transcription of template DNA in HeLa whole-cell extract does terminate near the 5' end of this sequence. Nuclease S1 and exonuclease VII mapping of the 3' region of the in vitro synthesized RNAs confirm these results. The termination activity of the sequence A9T2A5TA4TA4TA5 is independent of the distance from the promoter and of the nature of the DNA template (linear vs. circular). The termination activity of the sequence shows a strong orientation dependence. These results strongly suggest that the termination activity of this cis-acting element is modulated by a trans-acting cellular factor. The unique structural feature of this sequence, a 10.5-base-pair inverted repeat, may determine the specificity of interaction of the trans-acting factor with the cis-acting element, resulting in accurate termination of transcription.
Recent advances in our understanding of transcription factors that regulate RNA polymerase II transcribed genes are described. This review considers the role of alternate protein-protein interactions to yield positive or negative regulation, the consequences of deregulated factor activity on cell function and approaches to establish factor-binding specificities.
During the development of multicellular organisms, the processes of growth and differentiation are kept in balance to generate and maintain tissues and organs of the correct size, shape and cellular composition. We have investigated the molecular controls of growth and differentiation in the Caenorhabditis elegans germline. A single somatic cell, called the distal tip cell, promotes mitotic proliferation in the adjacent germline by GLP-1/Notch signalling. Within the germline, the decisions between mitosis and meiosis and between spermatogenesis and oogenesis are controlled by a group of conserved RNA regulators. FBF, a member of the PUF (for Pumilio and FBF) family of RNA-binding proteins, promotes mitosis by repressing gld-1 mRNA activity; the GLD-1, GLD-2, GLD-3 and NOS-3 proteins promote entry into meiosis by regulating mRNAs that remain unknown. The regulatory balance between opposing FBF and GLD activities is crucial for controlling the extent of germline proliferation. PUF proteins regulate germline stem cells in both Drosophila and C. elegans and are localized to germline stem cells of the mammalian testis. Therefore, this post-transcriptional regulatory switch may be an ancient mechanism for controlling maintenance of stem cells versus differentiation.
Mutations in RNA-binding proteins are increasingly identified in cancers through tumor sequencing and are correlated with disease progression, therapy response, and overall patient outcomes, underscoring the need to study them. Here, we focus on the RNA-binding protein Poly-C binding protein 1 (PCBP1), which binds target RNAs through K-homology (KH) domains to regulate RNA fate. PCBP1 is a tumor suppressor gene and hotspot missense mutations at leucine residues 100 and 102 are observed in colorectal cancer (CRC). PCBP1 mutations have been recurrently reported in CRC genome-wide mutation studies and are associated with poor clinical outcomes; however, their effects on PCBP1 expression and function remain largely unexplored. We show that cancer-associated mutations substituting leucine 100 and 102 with glutamine, proline, or arginine destabilize PCBP1, leading to increased protein turnover. The L100/L102 residues occur at the interface of the RNA-binding KH1 and KH2 domains, and our molecular dynamics simulations show that mutations at these residues disrupt the secondary structure of PCBP1. Additionally, these mutants display increased cytoplasmic localization. Importantly, mutant PCBP1 physically interacts with wild type PCBP1 and suppresses its expression through a dominant-negative mechanism. Together, our data demonstrate that CRC-associated PCBP1 mutations destabilize the protein and act as dominant-negative variants, revealing a novel mechanism of tumor suppressor inactivation in colorectal cancer.