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PubMed2001

Purification of Drosophila snRNPs and characterization of two populations of functional U1 particles.

U1 snRNP is required at an early stage during assembly of the spliceosome, the dynamic ribonucleoprotein (RNP) complex that performs nuclear pre-mRNA splicing. Here, we report the purification of U1 snRNP particles from Drosophila nuclear extracts and the characterization of their biochemical properties, polypeptide contents, and splicing activities. On the basis of their antigenicity, apparent molecular weight, and by peptide sequencing, the Drosophila 70K, SNF, B, U1-C, D1, D2, D3, E, F, and G proteins are shown to be integral components of these particles. Sequence database searches revealed that both the U1-specific and the Sm proteins are extensively conserved between human and Drosophila snRNPs. Furthermore, both species possess a conserved intrinsic U1-associated kinase activity with identical substrate specificity in vitro. Finally, our results demonstrate that a second type of functional U1 particle, completely lacking the U1/U2-specific protein SNF and the associated protein kinase activity, can be isolated from cultured Kc cell or Canton S embryonic nuclear extracts. This work describes the first characterization of a purified Drosophila snRNP particle and reinforces the view that their activity and composition, with the exception of the atypical bifunctional U1-A/U2-B" SNF protein, are highly conserved in metazoans.

Amino Acid Sequence↗
PubMed2001

Computational modeling of eukaryotic mRNA turnover.

The process of eukaryotic gene expression involves a diverse number of steps including transcription, RNA processing, transport, translation, and mRNA turnover. A critical step in understanding this process will be the development of mathematical models that quantitatively describe and predict the behavior of this complex system. We have simulated eukaryotic mRNA turnover in a linear multicomponent model based on the known mRNA decay pathways in yeast. Using rate constants based on experimental data for the yeast unstable MFA2 and stable PGK1 transcripts, the computational modeling reproduces experimental observations after minor adjustments. Subsequent analysis and a series of in silico experiments led to several conclusions. First, we demonstrate that mRNA half-life as commonly measured underestimates the average life span of an mRNA. Second, due to the properties of the pathways, the measurement of a half-life can predominantly measure different steps in the decay network. A corollary of this fact is that different mRNAs will be affected differentially by changes in specific rate constants. Third, the way to obtain the largest change of levels of mRNA for the smallest changes in rate is by changing the rate of deadenylation, where a large amount of regulation of mRNA decay occurs. Fourth, the 3'-to-5' degradation of mRNA shows mRNA-specific rates of degradation that are dependent on the 5' structure of the mRNA. These programs can be run over the Web, are adaptable to other eukaryotes, and provide outputs as graphs and virtual northern gels, which can be directly compared to experimental data. Therefore, this model constitutes a useful tool for the quantitative analysis of the process and control of mRNA degradation in eukaryotic cells.

Biological Transport↗
PubMed2002

Defining a 5' splice site by functional selection in the presence and absence of U1 snRNA 5' end.

Pre-mRNA splicing in metazoans is mainly specified by sequences at the termini of introns. We have selected functional 5' splice sites from randomized intron sequences through repetitive rounds of in vitro splicing in HeLa cell nuclear extract. The consensus sequence obtained after one round of selection in normal extract closely resembled the consensus of natural occurring 5' splice sites, suggesting that the selection pressures in vitro and in vivo are similar. After three rounds of selection under competitive splicing conditions, the base pairing potential to the U1 snRNA increased, yielding a G100%U100%R94%A67%G89%U76%R83% intronic consensus sequence. Surprisingly, a nearly identical consensus sequence was obtained when the selection was performed in nuclear extract containing U1 snRNA with a deleted 5' end, suggesting that other factors than the U1 snRNA are involved in 5' splice site recognition. The importance of a consecutive complementarity between the 5' splice site and the U1 snRNA was analyzed systematically in the natural range for in vitro splicing efficiency and complex formation. Extended complementarity was inhibitory to splicing at a late step in spliceosome assembly when pre-mRNA substrates were incubated in normal extract, but favorable for splicing under competitive splicing conditions or in the presence of truncated U1 snRNA where transition from complex A to complex B occurred more rapidly. This suggests that stable U1 snRNA binding is advantageous for assembly of commitment complexes, but inhibitory for the entry of the U4/U6.U5 tri-snRNP, probably due to a delayed release of the U1 snRNP.

3' Untranslated Regions↗
PubMed1999

The cis acting sequences responsible for the differential decay of the unstable MFA2 and stable PGK1 transcripts in yeast include the context of the translational start codon.

A general pathway of mRNA turnover has been described for yeast in which the 3' poly(A) tail is first deadenylated to an oligo(A) length, leading to decapping and subsequent 5'-3' exonucleolytic decay. The unstable MFA2 mRNA and the stable PGK1 mRNAs both decay through this pathway, albeit at different rates of deadenylation and decapping. To determine the regions of the mRNAs that are responsible for these differences, we examined the decay of chimeric mRNAs derived from the 5' untranslated, coding, and 3' untranslated regions of these two mRNAs. These experiments have led to the identification of the features of these mRNAs that lead to their different stabilities. The MFA2 mRNA is unstable solely because its 3' UTR promotes the rates of deadenylation and decapping; all other features of this mRNA are neutral with respect to mRNA decay rates. The PGK1 mRNA is stable because the sequence context of the PGK1 translation start codon and the coding region function together to stabilize the transcript, whereas the PGK13' UTR is neutral with respect to decay. Importantly, changes in the PGK1 start codon context that destabilized the transcript also reduced its translational efficiency. This observation suggests that the nature of the translation initiation complex modulates the rates of mRNA decapping and decay.

3' Untranslated Regions↗
PubMed2001

RNA-catalyzed amino acid activation.

We have selected RNAs that perform a new reaction that chemically activates amino acids, paralleling mixed phosphate anhydride synthesis by protein aminoacyl-transfer RNA synthetases. Care with recovery of the unstable reaction product was apparently essential to this selection. The best characterized RNA, KK13, requires only Ca2+ for reaction and is optimally active at low pH with KM = 50 mM and kcat = 1.1 min(-1) for activation of leucine. In conjunction with previous RNA-catalyzed aminoacyl-RNA synthesis, peptide bond formation, and RNA-based coding, these amino acid-activating RNAs complete an experimental demonstration that the four fundamental reactions of protein biosynthesis can be RNA-mediated. The appearance of translation in an RNA world is therefore supported.

Amino Acids↗
PubMed2000

Wheat germ translation initiation factor eIF4B affects eIF4A and eIFiso4F helicase activity by increasing the ATP binding affinity of eIF4A.

It has been proposed that, during translational initiation, structures in the 5' untranslated region of mRNA are unwound. eIF4A, a member of the DEAD box family of proteins (those that contain a DEAD amino acid sequence), separately or in conjunction with other eukaryotic initiation factors, utilizes the energy from ATP hydrolysis to unwind these structures. As a step in defining the mechanism of helicase activity in the wheat germ protein synthesis system, we have utilized direct fluorescence measurements, ATPase assays, and helicase assays. The RNA duplex unwinding activity of wheat germ eIF4A is similar to other mammalian systems; however, eIF4F or eIFiso4F is required, probably because of the low binding affinity of wheat germ eIF4A for mRNA. Direct ATP binding measurements showed that eIF4A had a higher binding affinity for ADP than ATP, resulting in a limited hydrolysis and procession along the RNA in the helicase assay. The addition of eIF4B resulted in a change in binding affinity for ATP, increasing it almost 10-fold while the ADP binding affinity was approximately the same. The data presented in this paper suggest that eIF4F or eIFiso4F acts to position the eIF4A and stabilize the interaction with mRNA. ATP produces a conformational change which allows a limited unwinding of the RNA duplex. The binding of eIF4B either prior to or after hydrolysis allows for increased affinity for ATP and for the cycle of conformational changes to proceed, resulting in further unwinding and processive movement along the mRNA.

Adenosine Diphosphate↗
PubMed1997

Transcription initiation sites for sorghum mitochondrial atp9 are positioned immediately 3' to trnfM.

Sorghum mitochondrial atp9 is polymorphic among male-sterile cytoplasms, but each cytoplasm is characterized by a major 650 nt transcript, regardless of fertility status. The gene is positioned 323 bp 3' to trnfM. Primer extension revealed multiple atp9 5' transcript termini, distributed from +1 to +28 3' to trnfM; the termini could be labeled with polynucleotide kinase, suggesting that they result from the maturation of trnfM. Guanylyltransferase experiments, however, showed that four of the termini were capable. The juxtaposition of a putative promoter 3' to trnfM results in a unique atp9 transcript population consisting of primary and processed transcripts.

Amino Acid Sequence↗
PubMed1999

Internal ribosome entry sites (IRESs): reality and use.

IRESs are known to recruit ribosomes directly, without a previous scanning of untranslated region of mRNA by the ribosomes. IRESs have been found in a number of viral and cellular mRNAs. Experimentally, IRESs are commonly used to direct the expression of the second cistrons of bicistronic mRNAs. The mechanism of action of IRESs is not fully understood and a certain number of laboratories were not successful in using them in a reliable manner. Three observations done in our laboratory suggested that IRESs might not work as functionally as it was generally believed. Stem loops added before IRESs inhibited mRNA translation. When added into bicistronic mRNAs, IRESs initiated translation of the second cistrons efficiently only when the intercistronic region contained about 80 nucleotides, and they did not work any more effectively with intercistronic regions containing at least 300-400 nucleotides. Conversely, IRESs inserted at any position into the coding region of a cistron interrupted its translation and initiated translation of the following cistron. The first two data are hardly compatible with the idea that IRESs are able to recruit ribosomes without using the classical scanning mechanism. IRESs are highly structured and cannot be scanned by the 40S ribosomal subunit. We suggest that IRESs are short-circuited and are essentially potent stimulators favoring translation in particular physiological situations.

Animals↗
PubMedDate not supplied

Light regulation of plant gene expression by an upstream enhancer-like element.

Light regulates many varied physiological and developmental phenomena during plant growth and differentiation, including the formation of a photosynthetically competent chloroplast from a proplastid. The expression of ribulose 1,5-bisphosphate carboxylase small subunit (rbcS) genes is regulated by light in a development- and tissue-specific manner2,3. In some plant species, phytochrome has been demonstrated to mediate this response, and photoregulation of rbcS expression occurs at least in part at the level of transcription. We have shown previously that a 5'-noncoding fragment (4-973 base pairs (bp) upstream of the messenger RNA cap site) of the pea rbcS ss3.6 gene contains all of the nucleotide sequence information necessary to direct the photoregulated expression of a bacterial chloramphenicol acetyltransferase (cat) gene in tobacco. Consistent with these findings, Morelli et al.11 have shown by deletion analysis of a second rbcS gene promoter, that the sequences required for photoregulated expression of rbcS E9 reside within the 5'-noncoding region. They identified an upstream region of approximately 700 bp needed for maximum transcription but not light-dark regulation, and a region from -35 to -2 bp which included the TATA box and contained the necessary information for light responsiveness. We now demonstrate that regulatory sequences 5' distal to the rbcS ss3.6 TATA box and transcriptional start site not only contain the information necessary for maximum expression, but also confer photoregulation. These upstream regulatory sequences function independently of orientation when fused to their homologous promoter or a heterologous promoter.

Acetyltransferases↗
PubMed2002

An extensive network of coupling among gene expression machines.

Gene expression in eukaryotes requires several multi-component cellular machines. Each machine carries out a separate step in the gene expression pathway, which includes transcription, several pre-messenger RNA processing steps and the export of mature mRNA to the cytoplasm. Recent studies lead to the view that, in contrast to a simple linear assembly line, a complex and extensively coupled network has evolved to coordinate the activities of the gene expression machines. The extensive coupling is consistent with a model in which the machines are tethered to each other to form 'gene expression factories' that maximize the efficiency and specificity of each step in gene expression.

Gene Expression↗
PubMed2007

Illuminating the silence: understanding the structure and function of small RNAs.

RNA interference (RNAi) is triggered by double-stranded RNA helices that have been introduced exogenously into cells as small interfering (si)RNAs or that have been produced endogenously from small non-coding RNAs known as microRNAs (miRNAs). RNAi has become a standard experimental tool and its therapeutic potential is being aggressively harnessed. Understanding the structure and function of small RNAs, such as siRNAs and miRNAs, that trigger RNAi has shed light on the RNAi machinery. In particular, it has highlighted the assembly and function of the RNA-induced silencing complex (RISC), and has provided guidelines to efficiently silence genes for biological research and therapeutic applications of RNAi.

Animals↗
PubMed2006

Structural and mechanistic insights into hepatitis C viral translation initiation.

Hepatitis C virus uses an internal ribosome entry site (IRES) to control viral protein synthesis by directly recruiting ribosomes to the translation-start site in the viral mRNA. Structural insights coupled with biochemical studies have revealed that the IRES substitutes for the activities of translation-initiation factors by binding and inducing conformational changes in the 40S ribosomal subunit. Direct interactions of the IRES with initiation factor eIF3 are also crucial for efficient translation initiation, providing clues to the role of eIF3 in protein synthesis.

5' Untranslated Regions↗
PubMed1998

RNA chiropractics.

Explore the source record for details and available documents.

Binding Sites↗
PubMed2005

The yeast EDC1 mRNA undergoes deadenylation-independent decapping stimulated by Not2p, Not4p, and Not5p.

A major mechanism of eukaryotic mRNA degradation initiates with deadenylation followed by decapping and 5' to 3' degradation. We demonstrate that the yeast EDC1 mRNA, which encodes a protein that enhances decapping, has unique properties and is both protected from deadenylation and undergoes deadenylation-independent decapping. The 3' UTR of the EDC1 mRNA is sufficient for both protection from deadenylation and deadenylation-independent decapping and an extended poly(U) tract within the 3' UTR is required. These observations highlight the diverse forms of decapping regulation and identify a feedback loop that can compensate for decreases in activity of the decapping enzyme. Surprisingly, the decapping of the EDC1 mRNA is slowed by the loss of Not2p, Not4p, and Not5p, which interact with the Ccr4p/Pop2p deadenylase complex. This indicates that the Not proteins can affect decapping, which suggests a possible link between the mRNA deadenylation and decapping machinery.

3' Untranslated Regions↗