Widespread use of TATA elements in the core promoters for RNA polymerases III, II, and I in fission yeast.

In addition to directing transcription initiation, core promoters integrate input from distal regulatory elements. Except for rare exceptions, it has been generally found that eukaryotic tRNA and rRNA genes do not contain TATA promoter elements and instead use protein-protein interactions to bring the TATA-binding protein (TBP), to the core promoter. Genomewide analysis revealed TATA elements in the core promoters of tRNA and 5S rRNA (Pol III), U1 to U5 snRNA (Pol II), and 37S rRNA (Pol I) genes in Schizosaccharomyces pombe. Using tRNA-dependent suppression and other in vivo assays, as well as in vitro transcription, we demonstrated an obligatory requirement for upstream TATA elements for tRNA and 5S rRNA expression in S. pombe. The Pol III initiation factor Brf is found in complexes with TFIIIC and Pol III in S. pombe, while TBP is not, consistent with independent recruitment of TBP by TATA. Template commitment assays are consistent with this and confirm that the mechanisms of transcription complex assembly and initiation by Pol III in S. pombe differ substantially from those in other model organisms. The results were extended to large-rRNA synthesis, as mutation of the TATA element in the Pol I promoter also abolishes rRNA expression in fission yeast. A survey of other organisms' genomes reveals that a substantial number of eukaryotes may use widespread TATAs for transcription. These results indicate the presence of TATA-unified transcription systems in contemporary eukaryotes and provide insight into the residual need for TBP by all three Pols in other eukaryotes despite a lack of TATA elements in their promoters.

Comparison of the RNA polymerase III transcription machinery in Schizosaccharomyces pombe, Saccharomyces cerevisiae and human.

Multi-subunit transcription factors (TF) direct RNA polymerase (pol) III to synthesize a variety of essential small transcripts such as tRNAs, 5S rRNA and U6 snRNA. Use by pol III of both TATA-less and TATA-containing promoters, together with progress in the Saccharomyces cerevisiae and human systems towards elucidating the mechanisms of actions of the pol III TFs, provides a paradigm for eukaryotic gene transcription. Human and S.cerevisiae pol III components reveal good general agreement in the arrangement of orthologous TFs that are distributed along tRNA gene control elements, beginning upstream of the transcription initiation site and extending through the 3' terminator element, although some TF subunits have diverged beyond recognition. For this review we have surveyed the Schizosaccharomyces pombe database and identified 26 subunits of pol III and associated TFs that would appear to represent the complete core set of the pol III machinery. We also compile data that indicate in vivo expression and/or function of 18 of the fission yeast proteins. A high degree of homology occurs in pol III, TFIIIB, TFIIIA and the three initiation-related subunits of TFIIIC that are associated with the proximal promoter element, while markedly less homology is apparent in the downstream TFIIIC subunits. The idea that the divergence in downstream TFIIIC subunits is associated with differences in pol III termination-related mechanisms that have been noted in the yeast and human systems but not reviewed previously is also considered.

Construction of FLAG and histidine tagging vectors for Schizosaccharomyces pombe.

Schizosaccharomyces pombe is becoming an increasingly popular model system for investigating important cellular processes. To facilitate detection, purification and functional studies of Sz. pombe gene products, we constructed two tagging expression vectors for use in Sz. pombe. These vectors allow proteins to be expressed ectopically as fusion proteins with a FLAG epitope and six histidine residue tags attached to their N-terminus or C-terminus. The function and applicability of these vectors were examined and the results are shown using the N-terminal tagging vector encoding Sfc6p, a subunit of the Sz. pombe RNA polymerase III general transcription factor, TFIIIC.

Control of transfer RNA maturation by phosphorylation of the human La antigen on serine 366.

Conversion of a nascent precursor tRNA to a mature functional species is a multipartite process that involves the sequential actions of several processing and modifying enzymes. La is the first protein to interact with pre-tRNAs in eukaryotes. An opal suppressor tRNA served as a functional probe to examine the activities of yeast and human (h)La proteins in this process in fission yeast. An RNA recognition motif and Walker motif in the metazoan-specific C-terminal domain (CTD) of hLa maintain pre-tRNA in an unprocessed state by blocking the 5'-processing site, impeding an early step in the pathway. Faithful phosphorylation of hLa on serine 366 reverses this block and promotes tRNA maturation. The results suggest that regulation of tRNA maturation at the level of RNase P cleavage may occur via phosphorylation of serine 366 of hLa.

Isolation and cloning of four subunits of a fission yeast TFIIIC complex that includes an ortholog of the human regulatory protein TFIIICbeta.

Eukaryotic tRNA genes are controlled by proximal and downstream elements that direct transcription by RNA polymerase (pol) III. Transcription factors (TFs) that reside near the initiation site are related in Saccharomyces cerevisiae and humans, while those that reside at or downstream of the B box share no recognizable sequence relatedness. Human TFIIICbeta is a transcriptional regulator that exhibits no homology to S. cerevisiae sequences on its own. We cloned an essential Schizosaccharomyces pombe gene that encodes a protein, Sfc6p, with homology to the S. cerevisiae TFIIIC subunit, TFC6p, that extends to human TFIIICbeta. We also isolated and cloned S. pombe homologs of three other TFIIIC subunits, Sfc3p, Sfc4p, and Sfc1p, the latter two of which are conserved from S. cerevisiae to humans, while the former shares homology with the S. cerevisiae B box-binding homolog only. Sfc6p is a component of a sequence-specific DNA-binding complex that also contains the B box-binding homolog, Sfc3p. Immunoprecipitation of Sfc3p further revealed that Sfc1p, Sfc3p, Sfc4p, and Sfc6p are associated in vivo and that the isolated Sfc3p complex is active for pol III-mediated transcription of a S. pombe tRNA gene in vitro. These results establish a link between the downstream pol III TFs in yeast and humans.

Transcription termination by RNA polymerase III in fission yeast. A genetic and biochemically tractable model system.

In order for RNA polymerase (pol) III to produce a sufficient quantity of RNAs of appropriate structure, initiation, termination, and reinitiation must be accurate and efficient. Termination-associated factors have been shown to facilitate reinitiation and regulate transcription in some species. Suppressor tRNA genes that differ in the dT(n) termination signal were examined for function in Schizosaccharomyces pombe. We also developed an S. pombe extract that is active for tRNA transcription that is described here for the first time. The ability of this tRNA gene to be transcribed in extracts from different species allowed us to compare termination in three model systems. Although human pol III terminates efficiently at 4 dTs and S. pombe at 5 dTs, Saccharomyces cerevisiae pol III requires 6 dTs to direct comparable but lower termination efficiency and also appears qualitatively distinct. Interestingly, this pattern of sensitivity to a minimal dT(n) termination signal was found to correlate with the sensitivity to alpha-amanitin, as S. pombe was intermediate between human and S. cerevisiae pols III. The results establish that the pols III of S. cerevisiae, S. pombe, and human exhibit distinctive properties and that termination occurs in S. pombe in a manner that is functionally more similar to human than is S. cerevisiae.

Terminator-specific recycling of a B1-Alu transcription complex by RNA polymerase III is mediated by the RNA terminus-binding protein La.

Efficient synthesis of many small abundant RNAs is achieved by the proficient recycling of RNA polymerase (pol) III and stable transcription complexes. Cellular Alu and related retroposons represent unusual pol III genes that are normally repressed but are activated by viral infection and other conditions. The core sequences of these elements contain pol III promoters but must rely on fortuitous downstream oligo(dT) tracts for terminator function. We show that a B1-Alu gene differs markedly from a classical pol III gene (tRNAiMet) in terminator sequence requirements. B1-Alu genes that differ only in terminator sequence context direct differential RNA 3' end formation. These genes are assembled into stable transcription complexes but differ in their ability to be recycled in the presence of the La transcription termination factor. La binds to the nascent RNA 3' UUUOH end motif that is generated by transcriptional termination within the pol III termination signal, oligo(dT). We found that the recycling efficiency of the B1-Alu genes is correlated with the ability of La to access the 3' end of the nascent transcript and protect it from 3'-5' exonucleolytic processing. These results illuminate a relationship between RNA 3' end formation and transcription termination, and La-mediated reinitiation by pol III.

5' processing of tRNA precursors can Be modulated by the human La antigen phosphoprotein.

Eukaryotic precursor (pre)-tRNAs are processed at both ends prior to maturation. Pre-tRNAs and other nascent transcripts synthesized by RNA polymerase III are bound at their 3' ends at the sequence motif UUUOH [3' oligo(U)] by the La antigen, a conserved phosphoprotein whose role in RNA processing has been associated previously with 3'-end maturation only. We show that in addition to its role in tRNA 3'-end maturation, human La protein can also modulate 5' processing of pre-tRNAs. Both the La antigen's N-terminal RNA-binding domain and its C-terminal basic region are required for attenuation of pre-tRNA 5' processing. RNA binding and nuclease protection assays with a variety of pre-tRNA substrates and mutant La proteins indicate that 5' protection is a highly selective activity of La. This activity is dependent on 3' oligo(U) in the pre-tRNA for interaction with the N-terminal RNA binding domain of La and interaction of the C-terminal basic region of La with the 5' triphosphate end of nascent pre-tRNA. Phosphorylation of La is known to occur on serine 366, adjacent to the C-terminal basic region. We show that this modification interferes with the La antigen's ability to protect pre-tRNAiMet from 5' processing either by HeLa extract or purified RNase P but that it does not affect interaction with the 3' end of pre-tRNA. These findings provide the first evidence to indicate that tRNA 5'-end maturation may be regulated in eukaryotes. Implications of triphosphate recognition is discussed as is a role for La phosphoprotein in controlling transcriptional and posttranscriptional events in the biogenesis of polymerase III transcripts.

Heterodimer SRP9/14 is an integral part of the neural BC200 RNP in primate brain.

BC200 RNA is a brain-specific, small non-messenger RNA with a somatodendritic localization in primate neurons and a constituent of a ribonucleoprotein (RNP) complex. The primary and secondary structure of the 5' domain of BC200 RNA resembles that of the Alu domain of 7SL RNA, which is an integral part of the signal recognition particle (SRP). This would predict that similar proteins bind to this defined domain of both RNA species in vitro and in vivo. The data presented in this paper reveal that a protein that binds BC200 RNA in vivo is immunoreactive with antibodies against SRP9. This further supports the notion that the 5' domain of the BC200 RNA can fold into structures similar to the SRP Alu domain and, as a result, bind identical or similar proteins in vivo. The SRP9 protein binds only as dimer with SRP14 protein to the Alu domain of 7SL RNA to form a subdomain that, in SRP, is functional in translation arrest. Therefore, our data also indicate that the neuronal BC200 RNP is a candidate for regulating decentralized protein biosynthesis in dendrites, possibly with a mechanism that resembles translation arrest of the SRP.

A carboxy-terminal basic region controls RNA polymerase III transcription factor activity of human La protein.

Human La protein has been shown to serve as a transcription factor for RNA polymerase III (pol III) by facilitating transcription termination and recycling of transcription complexes. In addition, La binds to the 3' oligo(U) ends common to all nascent pol III transcripts, and in the case of B1-Alu RNA, protects it from 3'-end processing (R. J. Maraia, D. J. Kenan, and J. D. Keene, Mol. Cell. Biol. 14:2147-2158, 1994). Others have previously dissected the La protein into an N-terminal domain that binds RNA and a C-terminal domain that does not. Here, deletion and substitution mutants of La were examined for general RNA binding, RNA 3'-end protection, and transcription factor activity. Although some La mutants altered in a C-terminal basic region bind RNA in mobility shift assays, they are defective in RNA 3'-end protection and do not support transcription, while one C-terminal substitution mutant is defective only in transcription. Moreover, a C-terminal fragment lacking RNA binding activity appears able to support low levels of transcription by pol III. While efficient multiround transcription is supported only by mutants that bind RNA and contain a C-terminal basic region. These analyses indicate that RNA binding contributes to but is not sufficient for La transcription factor activity and that the C-terminal domain plays a role in transcription that is distinguishable from simple RNA binding. The transcription factor activity of La can be reversibly inhibited by RNA, suggesting the potential for feedback inhibition of pol III transcription.

The decline in human Alu retroposition was accompanied by an asymmetric decrease in SRP9/14 binding to dimeric Alu RNA and increased expression of small cytoplasmic Alu RNA.

Alu interspersed elements are inserted into the genome by a retroposition process that occurs via dimeric Alu RNA and causes genetic disorders in humans. Alu RNA is labile and can be diverted to a stable left monomer transcript known as small cytoplasmic Alu (scAlu) RNA by RNA 3' processing, although the relationship between Alu RNA stability, scAlu RNA production, and retroposition has been unknown. In vivo, Alu and scAlu transcripts interact with the Alu RNA-binding subunit of signal recognition particle (SRP) known as SRP9/14. We examined RNAs corresponding to Alu sequences that were differentially active during primate evolution, as well as an Alu RNA sequence that is currently active in humans. Mutations that accompanied Alu RNA evolution led to changes in a conserved structural motif also found in SRP RNAs that are associated with thermodynamic destabilization and decreased affinity of the Alu right monomer for SRP9/14. In contrast to the right monomer, the Alu left monomer maintained structural integrity and high affinity for SRP9/14, indicating that scAlu RNA has been under selection during human evolution. Loss of Alu right monomer affinity for SRP9/14 is associated with scAlu RNA production from Alu elements in vivo. Moreover, the loss in affinity coincided with decreased rates of Alu amplification during primate evolution. This indicates that stability of the Alu right monomer is a critical determinant of Alu retroposition. These results provide insight into Alu mobility and evolution and into how retroposons may interact with host proteins during genome evolution.

Phosphorylation of the human La antigen on serine 366 can regulate recycling of RNA polymerase III transcription complexes.

The human La antigen is an RNA-binding protein that facilitates transcriptional termination and reinitiation by RNA polymerase III. Native La protein fractionates into transcriptionally active and inactive forms that are unphosphorylated and phosphorylated at serine 366, respectively, as determined by enzymatic and mass spectrometric analyses. Serine 366 comprises a casein kinase II phosphorylation site that resides within a conserved region in the La proteins from several species. RNA synthesis from isolated transcription complexes is inhibited by casein kinase II-mediated phosphorylation of La serine 366 and is reversible by dephosphorylation. This work demonstrates a novel mechanism of transcriptional control at the level of recycling of stable transcription complexes.

A highly conserved nucleotide in the Alu domain of SRP RNA mediates translation arrest through high affinity binding to SRP9/14.

Binding of the signal recognition particle (SRP) to signal sequences during translation leads to an inhibition of polypeptide elongation known as translation arrest. The arrest activity is mediated by a discrete domain comprised of the Alu portion of SRP RNA and a 9 and 14 kDa polypeptide heterodimer (SRP9/14). Although very few nucleotides in SRP RNA are conserved throughout evolution, the remarkable conservation of G24, which resides in the region of SRP9/14 interaction, suggests that it is essential for translation arrest. To understand the functional significance of the G24 residue, we made single base substitutions in SRP RNA at this position and analyzed the ability of the mutants to bind SRP9/14 and to reconstitute functional SRPs. Mutation of G24 to C reduced binding to SRP9/14 by at least 50-fold, whereas mutation to A and U reduced binding approximately 2- and 5-fold respectively. The mutant RNAs could nevertheless assemble into SRPs at high subunit concentrations. SRPs reconstituted with mutant RNAs were not significantly defective in translation arrest assays, indicating that the conserved guanosine does not interact directly with the translational machinery. Taken together, these results demonstrate that G24 plays an important role in the translation arrest function of SRP by mediating high affinity binding of SRP9/14.

Monomeric scAlu and nascent dimeric Alu RNAs induced by adenovirus are assembled into SRP9/14-containing RNPs in HeLa cells.

Nearly 1 000 000 copies of Alu interspersed elements comprise approximately 5% of human DNA. Alu elements cause gene disruptions by a process known as retrotransposition, in which dimeric Alu RNA is a presumed intermediate. Dimeric Alu transcripts are labile, giving rise to stable left monomeric scAlu RNAs whose levels are tightly regulated. Induction of Alu RNA by viral infection or cell stress leads to a dramatic increase in dimeric Alu transcripts, while scAlu RNA increases modestly. Each monomer of the dimeric Alu element shares sequence homology with the 7SL RNA component of the signal recognition particle (SRP). The SRP protein known as SRP9/14 is also found in a discrete complex with scAlu RNA, although whether dimeric Alu RNA is associated with SRP9/14 had been unknown. Here we show that antiserum to human SRP9 immunoprecipitates both scAlu RNA and dimeric Alu RNAs and that these RNPs accumulate after adenovirus infection, while levels of SRP9, SRP14, SRP54 and 7SL SRP RNA are unaffected. Dimeric Alu RNAs are also associated with the La protein, indicating that these are indeed nascent RNA polymerase III transcripts. This report documents that induced Alu transcripts are assembled into SRP9/14-containing RNPs in vivo while SRP levels are unchanged. Implications for Alu RNA metabolism and evolution are discussed.

Transcription termination factor La is also an initiation factor for RNA polymerase III.

La RNA-binding protein is a transcription termination factor that facilitates recycling of template and RNA polymerase (pol) 111. Transcription complexes preassembled on immobilized templates were depleted of pol III after a single round of RNA synthesis in the presence of heparin and sarkosyl. The isolated complexes could then be complemented with highly purified pol III and/or recombinant La to test if La is required for transcription reinitiation. VA1, 7SL, and B1 transcription complexes cannot be transcribed by supplemental pol III in single or multiple-round transcription assays unless La is also provided. La mediates concentration-dependent activation of pol III initiation and thereby controls the use of preassembled stable transcription complexes. The initiation factor activity of La augments its termination factor activity to produce a novel mechanism of activated reinitiation. A model in which La serves pol III upon transcription initiation and again at termination is discussed.

Human signal recognition particle (SRP) Alu-associated protein also binds Alu interspersed repeat sequence RNAs. Characterization of human SRP9.

Nearly 1 million interspersed Alu elements reside in the human genome. Alu retrotransposition is presumably mediated by full-length Alu transcripts synthesized by RNA polymerase III, while some polymerase III-synthesized Alu transcripts undergo 3'-processing and accumulate as small cytoplasmic (sc) RNAs of unknown function. Interspersed Alu sequences also reside in the untranslated regions of some mRNAs. The Alu sequence is related to a portion of the 7SL RNA component of signal recognition particle (SRP). This region of 7SL RNA together with 9- and 14-kDa polypeptides (SRP9/14) regulates translational elongation of ribosomes engaged by SRP. Here we characterize human (h) SRP9 and show that it, together with hSRP14 (SRP9/14), forms the activity previously identified as Alu RNA-binding protein (RBP). The primate-specific C-terminal tail of hSRP14 does not appreciably affect binding to scAlu RNA. Kd values for three Alu-homologous scRNAs were determined using Alu RBP (SRP9/14) purified from HeLa cells. The Alu region of 7SL, scAlu, and scB1 RNAs exhibited Kd values of 203 pM, 318 pM, and 1.8 nM, respectively. Finally, Alu RBP can bind with high affinity to synthetic mRNAs that contain interspersed Alus in their untranslated regions.

A trinucleotide repeat-associated increase in the level of Alu RNA-binding protein occurred during the same period as the major Alu amplification that accompanied anthropoid evolution.

Nearly 1 million Alu elements in human DNA were inserted by an RNA-mediated retroposition-amplification process that clearly decelerated about 30 million years ago. Since then, Alu sequences have proliferated at a lower rate, including within the human genome, in which Alu mobility continues to generate genetic variability. Initially derived from 7SL RNA of the signal recognition particle (SRP), Alu became a dominant retroposon while retaining secondary structures found in 7SL RNA. We previously identified a human Alu RNA-binding protein as a homolog of the 14-kDa Alu-specific protein of SRP and have shown that its expression is associated with accumulation of 3'-processed Alu RNA. Here, we show that in early anthropoids, the gene encoding SRP14 Alu RNA-binding protein was duplicated and that SRP14-homologous sequences currently reside on different human chromosomes. In anthropoids, the active SRP14 gene acquired a GCA trinucleotide repeat in its 3'-coding region that produces SRP14 polypeptides with extended C-terminal tails. A C-->G substitution in this region converted the mouse sequence CCA GCA to GCA GCA in prosimians, which presumably predisposed this locus to GCA expansion in anthropoids and provides a model for other triplet expansions. Moreover, the presence of the trinucleotide repeat in SRP14 DNA and the corresponding C-terminal tail in SRP14 are associated with a significant increase in SRP14 polypeptide and Alu RNA-binding activity. These genetic events occurred during the period in which an acceleration in Alu retroposition was followed by a sharp deceleration, suggesting that Alu repeats coevolved with C-terminal variants of SRP14 in higher primates.

The human Y4 small cytoplasmic RNA gene is controlled by upstream elements and resides on chromosome 7 with all other hY scRNA genes.

Ro ribonucleoproteins (RNP) constitute a class of evolutionarily conserved small cytoplasmic (sc) RNPs whose functions are unknown. In human cells four distinctive scRNAs designated hY1, hY3, hY4 and hY5 are synthesized by RNA polymerase III (pol III) and accumulate as components of Ro scRNPs. The previously isolated hY1 and hY3 genes contain upstream sequences similar to the class III promoters for U6 and 7SK snRNAs. Additional mammalian Y scRNA genes have been refractory to cloning due to interference from numerous hY-homologous pseudogenes and studies of hY RNA genes have been sparse. Although homologs of hY1 and hY3 RNAs exist in rodent cells, the smaller Y4 and Y5 RNAs do not which has allowed us to localize the hY4 scRNA gene to human chromosome 7 by assaying for its transcript in rodent X human somatic cell hybrids (SCH). A chromosome 7-enriched yeast artificial chromosome (YAC) library was then screened and the authentic hY4 sequence was isolated by strepavidin--biotin-mediated hybrid-selection followed by poly(dA)-tailing and hemispecific PCR. The region upstream of the hY4 sequence contains a TATAAAA motif centered at -26, a candidate proximal sequence element at -63, and three octamer-like sequences located between -260 and -200. hY4 RNA is readily detectable on Northern blots after transient transfection of the hY4 gene into mouse cells but not after transfection of a construct in which the 5' flanking region was deleted. SCHs and chromosome 7-enriched YACs were used to demonstrate that all four hY RNA genes reside on human chromosome 7.