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PubMed2006

Voltage-gated potassium channels: regulation by accessory subunits.

Voltage-gated potassium channels regulate cell membrane potential and excitability in neurons and other cell types. A precise control of neuronal action potential patterns underlies the basic functioning of the central and peripheral nervous system. This control relies on the adaptability of potassium channel activities. The functional diversity of potassium currents, however, far exceeds the considerable molecular diversity of this class of genes. Potassium current diversity contributes to the specificity of neuronal firing patterns and may be achieved by regulated transcription, RNA splicing, and posttranslational modifications. Another mechanism for regulation of potassium channel activity is through association with interacting proteins and accessory subunits. Here the authors highlight recent work that addresses this growing area of exploration and discuss areas of future investigation.

Action Potentials↗

PubMed1992

Modifications of the guanidine hydrochloride procedure for the extraction of RNA: isolation from a variety of tissues and adherent/nonadherent cell types.

In a previous report dealing with the guanidine hydrochloride protocol for the extraction of RNA from mouse peritoneal macrophages, we identified a major source of RNA-degrading activity and showed that its removal early in the extraction procedure resulted in a more dependable method for the recovery of high-quality RNA. This report extends these findings and demonstrates the general applicability of the technique to a variety of fresh or frozen adherent cell types, cell suspensions and tissues, further highlighting stages at which degradation is most likely to occur and how to avoid a variety of pitfalls associated with the extraction procedure.

Animals↗

PubMed2006

Human fertility protein PUMILIO2 interacts in vitro with testis mRNA encoding Cdc42 effector 3 (CEP3).

PUMILIO protein regulates translation of specific mRNAs in morphogenesis and in development of the germ-line of model organisms such as flies and worms. Given that a human homologue (PUMILIO2) was recently identified in the germ-line stem cells, the question was raised whether it regulates translation of fertility mRNAs similarly to Drosophila Pumilio. Here, we describe a candidate mRNA encoding Cdc42 effector protein 3 (CEP3), however, a function for this protein in reproduction has previously not been reported. We detected three CEP3 transcripts in the testis tissue including one which was highly expressed and testis specific by northern blotting. We found that CEP mRNA contains GUUGU (A) and AUUGUA (B) motifs (ABB) within the 3' untranslated region (3'UTR), which are also present in mRNA targets of Pumilio in Drosophila. Interaction of PUMILIO2 with the fragment of CEP3 transcript containing the ABB array was tested by mobility shift assay and we found that PUMILIO2 binds the 3' untranslated region of the CEP3 mRNA. These results support the hypothesis that CEP3 mRNA may be a target of PUMILIO2 protein in the human male gonad and be under translational control mediated by specific nucleotide motifs within the 3'UTR.

3' Untranslated Regions↗

PubMed2004

Aminoacyl-tRNA synthesis by pre-translational amino acid modification.

Aminoacyl-tRNAs (aa-tRNAs) are essential substrates for ribosomal translation, and are generally synthesized by aminoacyl-tRNA synthetases (aaRSs). It was expected earlier that every organism would contain a complete set of twenty aaRSs, one for each canonical amino acid. However, analysis of the many known genome sequences and biochemical studies revealed that most organisms lack asparaginyl- and glutaminyl-tRNA synthetases, and thus are unable to attach asparagine and glutamine directly onto their corresponding tRNA. Instead, a pretranslational amino acid modification is required to convert Asp-tRNA(Asn) and Glu-tRNA(Gln) to the correctly charged Asn-tRNA(Asn) and Gln-tRNA(Gln), respectively. This transamidation pathway of amide aa-tRNA synthesis is common in most bacteria and archaea. Unexpected results from biochemical, genetic and genomic studies showed that a large variety of different bacteria rely on tRNA-dependent transamidation for the formation of the amino acid asparagine. Pretranslational modifications are not restricted to asparagine and glutamine but are also found in the biosynthesis of some other aa-tRNAs, such as the initiator tRNA fmet-tRNA(Met)(i) and Sec-tRNA(Sec) specifying selenocysteine, the 21(st) cotranslationally inserted amino acid. tRNA-dependent amino acid modification is also involved in the generation of aminolevulinic acid, the first precursor for porphyrin biosynthesis in many organisms.

Amino Acids↗

PubMed1985

Regulation of expression of immunoglobulins M and D in murine B cells.

Immunoglobulins M (IgM) and D (IgD) are expressed in a coordinantly regulated and differentiation-dependent fashion on the surface of B lymphocytes. We have studied the role of DNA configuration of their linked constant region genes C mu and C delta, as well as their transcription and posttranscriptional processing, in the regulation of these changes. After rearrangement of variable region segments, IgD can be singularly expressed in plasmacytoma cells by a DNA deletion of the C mu gene that is mediated by illegitimate recombination. However, IgM and IgD are usually expressed jointly without further DNA rearrangement downstream of VDJ. In pre-B cells, C delta apparently is not transcribed before light-chain expression. However, in early neonates (2 days old), C delta is transcribed at approximately one-third the level of C mu even though IgD is not detectable on the cell surface. This same ratio of transcription is preserved in older neonates (12 days old), which express only modest quantities of IgD, and in mature resting B cells, which express far higher densities of cell surface IgD than IgM. On activation by mitogens, transcription of C mu is preferentially enhanced, but it is surprising that C delta transcription remains at the baseline level even though cytoplasmic delta mRNA is virtually undetectable. The apparent discrepancy in transcription and ultimate expression can be explained by further modifications of both the RNA and polypeptide chains. Collectively, our data show that the differential expression of IgM and IgD is regulated by complex mechanisms at several levels.

Aging↗

PubMed1981

Excess synthesis of viral mRNA 5-terminal oligonucleotides by reovirus transcriptase.

Short oligonucleotides corresponding to the 5'-terminal sequence of reovirus mRNAs were produced in vitro by virion-associated transcriptase activity. Both capped and uncapped oligonucleotides were synthesized in molar excess relative to mRNA. Yields of uncapped oligomers including ppG-C and ppG-C-U were severalfold greater than the homologous capped structures. In partial reaction mixtures that were nonsupportive for mRNA chain elongation, capped oligomer synthesis was increased. Similarly, oligonucleotide formation was differentially resistant in viral preparations that were inactivated with respect to mRNA synthesis by modification of the genome RNA by dimethyl sulfate alkylation or psoralen photoreaction. The results suggest that reovirus mRNA synthesis involves excessive initiation by reiterative transcription of promoter sites by the reovirus polymerase. Only a small fraction of the resulting oligonucleotides are capped and extended to form full length mRNAs during a subsequent elongation step which is apparently mediated by transcriptase molecules that escape the reiterative phase of transcription.

Alkylating Agents↗

PubMedDate not supplied

Analysis of promoter and androgen regulatory sequences required for optimal transcription of the rat androgen-binding protein gene.

The androgen-binding protein (ABP) gene P1 promoter directs cell-specific gene regulation of ABP secreted by Sertoli cells. A recent study using the mouse Sertoli cell line (MSC-1) with a luciferase reporter system demonstrated Sertoli cell-specific gene expression with 619 bp of P1 DNA. Furthermore, based on studies of the rat and human genes, several controversies developed over the promoter characteristics, including the promoter type, the transcription start site, and whether the gene is regulated directly by androgens. In this study, the answers to several of these controversies were deciphered using the MSC-1 cell model. The results of mutagenesis experiments were consistent with the presence of the major transcription start site at 36 bp upstream of the initiating Met residue. Modification of the initiator sequence at the start site reduced activity in MSC-1 and NIH3T3 fibroblast cells. Mutation of a putative modified TATA sequence or conversion to the consensus TATA sequence had no effect on activity. Modification of a consensus RNA splice sequence at the start site also had no effect on activity. Furthermore, a minor start site was localized 179 bp upstream of the major site using reverse transcriptase-polymerase chain reaction with various P1 primers (primer walking), primer extension, and cDNA cloning. RNA transcripts from the minor site contain an untranslated 5' exon but apparently encode the same protein as the major transcript. The effect of androgens on P1 expression was also investigated. Cotransfection experiments with pCMVAR, which encodes the androgen receptor, demonstrated that dihydrotestosterone had no effect on the activity in MSC-1 cells. Taken together, these experiments and previous studies indicate that the rat ABP promoter P1 is regulated at the major start site by an initiator element without a TATA sequence, and the gene appears not to be directly regulated by follicle-stimulating hormone or androgens.

3T3 Cells↗

PubMed1999

RNA editing.

The term RNA editing describes those molecular processes in which the information content is altered in an RNA molecule. To date such changes have been observed in tRNA. rRNA and mRNA molecules of eukaryotes, but not prokaryotes. The demonstration of RNA editing in prokaryotes may only be a matter of time, considering the range of species in which the various RNA editing processes have been found. RNA editing occurs in the nucleus, as well as in mitochondria and plastids, which are thought to have evolved from prokaryotic-like endosymbionts. Most of the RNA editing processes, however, appear to be evolutionarily recent acquisitions that arose independently. The diversity of RNA editing mechanisms includes nucleoside modifications such as C to U and A to I deaminations, as well as non-templated nucleotide additions and insertions. RNA editing in mRNAs effectively alters the amino acid sequence of the encoded protein so that it differs from that predicted by the genomic DNA sequence.

Amino Acid Sequence↗

PubMed2005

Precise and parallel characterization of coding polymorphisms, alternative splicing, and modifications in human proteins by mass spectrometry.

The human proteome is a highly complex extension of the genome wherein a single gene often produces distinct protein forms due to alternative splicing, RNA editing, polymorphisms, and posttranslational modifications. Such biological variation compounded by the high sequence identity within gene families currently overwhelms the complete and routine characterization of mammalian proteins by MS. A new data base of human proteins (and their possible variants) was created and searched using tandem mass spectrometric data from intact proteins. This first application of top down MS/MS to wild-type human proteins demonstrates both gene-specific identification and the unambiguous characterization of multifaceted mass shifts (Deltam values). Such Deltam values found from the precise identification of 45 protein forms from HeLa cells reveal 34 coding single nucleotide polymorphisms, two protein forms from alternative splicing, and 12 diverse modifications (not including simple N-terminal processing), including a previously unknown phosphorylation at 10% occupancy. Automated protein identification was achieved with a median expectation value of 10(-13) and often occurred simultaneously with dissection of diverse sources of protein variability as they occur in combination. Top down MS therefore has a bright future for enabling precise annotation of gene products expressed from the human genome by non-mass spectrometrists.

Alternative Splicing↗

PubMed2002

Combination of interferon alfa-2b and ribavirin in liver transplant recipients with histological recurrent hepatitis C.

Recurrent hepatitis C virus (HCV) infection is an important cause of fibrosis and cirrhosis after liver transplantation (LT), with histological recurrence developing in at least 50% of patients within the first year. The aim of this study is to assess the safety and efficacy of interferon alfa-2b plus ribavirin in treating histological recurrent HCV after LT. Since 1998, patients with HCV with significant histological recurrence (fibrosis >/= 3 and/or histological activity index >/= 5) or progressive cholestatic disease after LT were treated with interferon alfa-2b (3 million units subcutaneously three times weekly) plus ribavirin (800 to 1,000 mg/d) for 12 months. Immunosuppression was tapered to cyclosporine/FK506 monotherapy. HCV RNA was assessed at entry, week 24, end of treatment, and 6 months after therapy. The primary end point was loss of HCV RNA 6 months after therapy, whereas the secondary end point was histological response. Fifty-four patients met criteria for treatment and have completed follow-up. Patients were mainly men (71% men; mean age, 51 +/- 5 years) with genotype 1 infection (88%) and high viral load (mean HCV RNA, 38 +/- 9 mEq/mL). Dose modification was required in 72% of patients because of cytopenia or side effects. Intent-to-treat analysis showed that serum HCV RNA was undetectable in 19 patients (35%) week 24, 21 patients (38%) week 48, and 16 patients (30%) at the 6-month follow-up. Paired liver biopsy results (before and within 6 months after treatment) were available for 35 patients. Patients who achieved viral eradication had no significant progression of fibrosis after 1 year of therapy. In summary, combination therapy is a reasonable antiviral option for recurrent HCV infection for established post-LT hepatitis and appears to prevent histological progression of disease if viral eradication is successful.

Adult↗

PubMed1991

Comparison and modification of rRNA sequencing methods.

In this study, modification of two methods of RNA sequencing resulted in more definitive sequencing bands. In one method of sequencing, the bands of lane A and lane G sometimes were not clear. Modifications of this method by changing the concentrations of ddATP and ddGTP resulted in the bands of lane A and lane G becoming more readable. Although a second sequencing method was found to have clearer bands than the first method, and the bases immediately downstream from the primer binding site could be read by using r-32P-labeled primer, the bands on the top of lane A still were not clear. Modifications of this second method by changing the ddATP/dATP ratio resulted in the bands of lane A becoming much clearer.

Base Sequence↗

PubMed1998

Probing of the spliceosome with site-specifically derivatized 5' splice site RNA oligonucleotides.

We have developed a site-specific chemical modification technique to incorporate a photoreactive azidophenacyl (APA) group at designated internal positions along the RNA phosphodiester backbone. Using this technique, we have analyzed interactions of the 5' splice site (5'SS) RNA within the spliceosome. Several crosslinked products can be detected within complex B using the derivatized 5'SS RNAs, including U6 snRNA, hPrp8p, and 114-, 90-, 70-, 54-, and 27-kDa proteins. The 5'SS RNAs derivatized at intron positions +4 to +8 crosslink to U6 snRNA, confirming the previously reported pairing interaction between these sequences. hPrp8p and p70 are crosslinked to the 5'SS RNA when the APA is placed within the 5' exon. Finally, a set of unidentified proteins, including p114, p54, and p27, is detected with the 5'SS RNA derivatized at intron positions +4 to +8. Introduction of the bulky APA group near the 5'SS junction (positions -2 to +3) strongly interferes with complex B formation and thus no APA crosslinks are observed at these positions. Together with our earlier observation that hPrp8p crosslinks to the GU dinucleotide at the 5' end of the intron, these results suggest that the inhibitory effect of APA results from steric hindrance of the hPrp8p:5'SS interaction. Unexpectedly, thio-modifications within the region of the 5'SS RNA that is involved in base pairing to U6 snRNA strongly stimulate complex B formation.

Allosteric Site↗

PubMed1999

Thiol-containing RNA for the study of structure and function of ribozymes.

RNA performs multiple functions in cellular environments, such as transferring genetic information, catalyzing chemical reactions, and providing an integral component of ribonucleoprotein complexes involved in mRNA processing and translation. Many of these functions are poorly understood, mainly due to the lack of structural information. Because limited information has been obtained by physical and biophysical techniques, chemical and biochemical methods have been extensively used for studying RNA structure. This article outlines one such method which relies on site-specific incorporation of thiols into RNA. A brief overview of the methods for incorporation of thiols into RNA is followed by a detailed description of a procedure which utilizes postsynthetic modification of 2'-amino-containing RNA for incorporation of thiols. The use of thiol-containing RNA to form disulfide cross-links for the study of the structure and dynamics of ribozymes is subsequently described.

Isothiocyanates↗

PubMed1995

RNA-Binding Characteristics of a Ribonucleoprotein from Spinach Chloroplast.

A chloroplast (nuclear-encoded) RNA-binding protein (28RNP) was previously purified from spinach (Spinacia oleracea). This 28RNP was found to be the major RNA-binding protein co-purified during the isolation scheme of 3[prime] end RNA-processing activity of several chloroplastic genes. To learn more about the possible involvement of 28RNP in the 3[prime] end RNA-processing event, we investigated the RNA-binding properties and the location of the protein in the chloroplast. We found that recombinant Escherichia coliexpressed 28RNP binds with apparently the same affinity to every chloroplastic 3[prime] end RNA that was analyzed, as well as to RNAs derived from the 5[prime] end or the coding region of some chloroplastic genes. Differences in the RNA-binding affinities for some chloroplastic 3[prime] end RNAs were observed when the recombinant 28RNP was compared with the "native" 28RNP in the chloroplast-soluble protein extract. In addition, we found that the 28RNP is not associated with either thylakoid-bound or soluble polysomes in which a great portion of the chloroplast rRNA and mRNA are localized. These results suggest that the native 28RNP binds specifically to certain RNA molecules in the chloroplast in which other components (possibly proteins) and/or posttranslational modifications are involved in determining RNA-binding specificity of the 28RNP.

Journal Article↗

PubMed2003

Inducible systemic RNA silencing in Caenorhabditis elegans.

Introduction of double-stranded RNA (dsRNA) can elicit a gene-specific RNA interference response in a variety of organisms and cell types. In many cases, this response has a systemic character in that silencing of gene expression is observed in cells distal from the site of dsRNA delivery. The molecular mechanisms underlying the mobile nature of RNA silencing are unknown. For example, although cellular entry of dsRNA is possible, cellular exit of dsRNA from normal animal cells has not been directly observed. We provide evidence that transgenic strains of Caenorhabditis elegans transcribing dsRNA from a tissue-specific promoter do not exhibit comprehensive systemic RNA interference phenotypes. In these same animals, modifications of environmental conditions can result in more robust systemic RNA silencing. Additionally, we find that genetic mutations can influence the systemic character of RNA silencing in C. elegans and can separate mechanisms underlying systemic RNA silencing into tissue-specific components. These data suggest that trafficking of RNA silencing signals in C. elegans is regulated by specific physiological and genetic factors.

Animals↗

PubMed1997

Epigenetic silencing of RNA polymerase I transcription: a role for DNA methylation and histone modification in nucleolar dominance.

Nucleolar dominance is an epigenetic phenomenon that describes nucleolus formation around rRNA genes inherited from only one progenitor of an interspecific hybrid or allopolyploid. The phenomenon is widespread, occurring in plants, insects, amphibians, and mammals, yet its molecular basis remains unclear. We have demonstrated nucleolar dominance in three allotetraploids of the plant genus Brassica. In Brassica napus, accurately initiated pre-rRNA transcripts from one progenitor, Brassica rapa are detected readily, whereas transcripts from the approximately 3000 rRNA genes inherited from the other progenitor, Brassica oleracea, are undetectable. Nuclear run-on confirmed that dominance is controlled at the level of transcription. Growth of B. napus seedlings on 5-aza-2'-deoxycytidine to inhibit cytosine methylation caused the normally silent, under-dominant B. oleracea rRNA genes to become expressed to high levels. The histone deacetylase inhibitors sodium butyrate and trichostatin A also derepressed silent rRNA genes. These results reveal an enforcement mechanism for nucleolar dominance in which DNA methylation and histone modifications combine to regulate rRNA gene loci spanning tens of megabase pairs of DNA.

Azacitidine↗

PubMed1998

Posttranscriptional modifications in 16S and 23S rRNAs of the archaeal hyperthermophile Sulfolobus solfataricus.

Posttranscriptional modification is common to many types of RNA, but the majority of information concerning structure and function of modification is derived principally from tRNA. By contrast, less is known about modification in rRNA in spite of accumulating evidence for its direct participation in translation. The structural identities and approximate molar levels of modifications have been established for 16S and 23S rRNAs of the archaeal hyperthermophile Sulfolobus solfactaricus by using combined chromatography-mass spectrometry-based methods. Modification levels are exceptionally high for prokaryotic organisms, with approximately 38 modified sites in 16S rRNA and 50 in 23S rRNA for cells cultured at 75 degrees C, compared with 11 and 23 sites, respectively, in Escherichia coli. We structurally characterized 10 different modified nucleosides in 16S rRNA, 64% (24 residues) of which are methylated at O-2' of ribose, and 8 modified species in 23S rRNA, 86% (43 residues) of which are ribose methylated, a form of modification shown in earlier studies to enhance stability of the polynucleotide chain. From cultures grown at progressively higher temperatures, 60, 75, and 83 degrees C, a slight trend toward increased ribose methylation levels was observed, with greatest net changes over the 23 degrees C range shown for 2'-O-methyladenosine in 16S rRNA (21% increase) and for 2'-O-methylcytidine (24%) and 2'-O-methylguanosine (22%) in 23S rRNA. These findings are discussed in terms of the potential role of modification in stabilization of rRNA in the thermal environment.

Alkylation↗

PubMed2000

Effects of anticodon 2'-O-methylations on tRNA codon recognition in an Escherichia coli cell-free translation.

The methylation of 2'-hydroxyl groups is one of the most common posttranscriptional modifications of naturally occurring stable RNA molecules. Some tRNA species have a 2'-O-methyl nucleoside at the first position of the anticodon, and it was suggested that this modification stabilizes the codon-anticodon duplex. However, no tRNA species have been found to have the modification at the second or third position of the anticodon. In the present study, we measured the effects of anticodon 2'-O-methylation on the codon-reading efficiencies of the anticodon variants of the unmodified forms of Escherichia coli tRNA1(Ser), using a cell-free protein synthesis assay. The modification of C in the first position of the anticodon into 2'-O-methylcytidine increased the efficiency of reading the G-ending codon. On the other hand, the modifications of the second and/or third positions were detrimental to the codon-reading activity. Thus, 2'-hydroxyl groups at the second and third positions of the anticodon may have some role in the translation reaction, and this may be the reason why 2'-O-methyl nucleosides are not found in these positions within natural tRNA species.

Amino Acid Sequence↗