No abstract

Short interspersed repetitive elements (SINEs) are a type of retroposon, being members of a class of informational molecules that are amplified via cDNA intermediates and flow back into the host genome. In contrast to retroviruses and retrotransposons, SINEs do not encode the enzymes required for their amplification, such as reverse transcriptases, so they are presumed to borrow these enzymes from other sources. In the present study, we isolated a family of long interspersed repetitive elements (LINEs) from the turtle genome. The sequence of this family was found to be very similar to those of the avian CR1 family. To our surprise, the sequence at the 3 end of the LINE in the turtle genome was nearly identical to that of a family of tortoise SINEs. Since CR1-like LINEs are widespread in birds and in many other reptiles, including the turtle, and since the tortoise SINEs are only found in vertical-necked turtles, it seems possible that the sequence at the 3 end of the tortoise SINEs might have been generated by recombination with the CR1-like LINE in a common ancestor of vertical-necked turtles, after the divergence of side-necked turtles. We extended our observations to show that the 3-end sequences of families of several tRNA-derived SINEs, such as the salmonid HpaI family, the tobacco TS family, and the salmon SmaI family, might have originated from the respective LINEs. Since it appears reasonable that the recognition sites of LINEs for reverse transcriptase are located within their 3-end sequences, these results provide the basis for a general scheme for the mechanism by which SINEs might acquire retropositional activity. We propose here that tRNA-derived SINEs might have been generated by a recombination event in which a strong-stop DNA with a primer tRNA, which is an intermediate in the replication of certain retroviruses and long terminal repeat retrotransposons, was directly integrated at the 3 end of a LINE.The reverse flow of genetic information from RNA back into DNA is known as retroposition, and each transposed informational element is known as a retroposon (23,36). Highly repetitive elements in eukaryotic genomes (28, 29), such as short interspersed repetitive elements (SINEs), long interspersed repetitive elements (LINEs), and processed retropseudogenes, are all included in this category. Retroposition appears to be a major evolutionary force that has contributed to the maintenance of the remarkable fluidity of eukaryotic genomes (28,36).

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 ␣-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. RNA polymerase (pol)1 III is a multisubunit enzyme that is directed to initiate RNA synthesis by transcription factors (TF) that bind to gene promoter elements. pol III transcripts comprise a large variety of small nuclear and cytoplasmic RNAs (1). Although there is diversity in the promoter structures of pol III-transcribed genes, three classes are responsible for the synthesis of most cellular pol III transcripts, tRNAs, 5 S rRNA, and U6 small nuclear RNA (2). Each of these represent a gene class that utilizes a characteristic promoter structure and specific set of TFs (3). 5 S rRNA genes comprise class I and contain a principal internal promoter that is recognized by TFIIIA. Class 3 genes utilize upstream TATA elements and in metazoans an upstream element recognized by a distinct multisubunit TF (4). Class 2 genes are represented by tRNA genes, which use an internal promoter comprised of proximal box A and distal box B elements. Distinct subunits of TFIIIC bind to the A box, B box, and terminator element of the class 2 genes and facilitate the assembly of this class of preinitiation complexes (3, 5). For each gene class, TFIIIB (or related activity) binds just upstream of the start site of transcription, and this in turn serves as the initiation factor proper as it recruits pol III (Refs. 6 -8 and references therein). Subunits of TFIIIB as well as pol III have been conserved from Saccharomyces cerevisiae to human, as have two TFIIIC subunits that localize near the start site of transcription (9 -14). By contrast, the downstream TFIIIC subunits in these organisms reveal no recognizable sequence homology (12,15,16).Some evidence suggests that efficient transcription requires termination and associa...

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.Structural similarities shared by the RNA polymerases (Pols) of bacteria, archaea, and eukarya reflect a deep-rooted common ancestry of transcription systems in all organisms on earth. Archaea and eukaryotes exhibit greater similarity to each other in their Pol subunits, accessory transcription factors (TFs), and promoter elements than either does to bacteria (36). In eukaryotes, Pol I synthesizes large rRNA (35S to 45S, depending on the species); Pol II synthesizes mRNAs and some small nuclear (sn) RNAs, such as U1 to U5; and Pol III synthesizes mostly tRNAs and 5S rRNA, as well as U6 snRNA and a few other transcripts (53).The core promoter orchestrates polymerase recruitment, promoter activity, and response to regulatory input (59, 66). In eukaryotes, TATA promoter elements direct transcription by Pol II of a large subset of (but not all) protein-encoding genes, but often not the far fewer snRNA genes that are transcribed by Pol II. While TATA elements are found in a minute fraction of Pol III genes, they are generally not found in the core promoter regions of Pol I genes (53). Intriguingly, despite the lack of TATA promoter elements, Pols I, II, and III all require TATA-binding protein (TBP) for initiation (17). Archaea use widespread TATA-like promoters and a TBP ortholog to direct transcription by a single Pol of all gene types, those encoding tRNA, rRNA, and mRNAs (reviewed in reference 67). Or...

Non-long-terminal-repeat (non-LTR) retrotransposons amplify their copies by reverse transcribing mRNA from the 3 end, but the initial processes of reverse transcription are still unclear. We have shown that a telomere-specific non-LTR retrotransposon of the silkworm, SART1, requires the 3 untranslated region (3 UTR) for retrotransposition. With an in vivo retrotransposition assay, we identified several novel motifs within the 3 UTR involved in precise and efficient reverse transcription. Of 461 nucleotides (nt) of the 3 UTR, the central region, from nt 163 to nt 295, was essential for SART1 retrotransposition. Of five putative stem-loops formed in RNA for the SART1 3 UTR, the second stem-loop (nt 159 to 221) is included in this region. Loss of the 3 region (nt 296 to 461) in the 3 UTR and the poly(A) tract resulted in decreased and inaccurate reverse transcription, which starts mostly from several telomeric repeat-like GGUU sequences just downstream of the second stem-loop. These results suggest that short telomeric repeat-like sequences in the 3 UTR anneal to the bottom strand of (TTAGG) n repeats. We also demonstrated that the mRNA for green fluorescent protein (GFP) could be retrotransposed into telomeric repeats when the GFP coding region is fused with the SART1 3 UTR and SART1 open reading frame proteins are supplied in trans.Non-long-terminal-repeat (non-LTR) retrotransposons are endogenous mobile genetic elements that are widespread among the genome of eukaryotes. Non-LTR retrotransposons multiply their copies through reverse transcription of RNA intermediates with a self-encoding reverse transcriptase (RT). Non-LTR retrotransposons, some of which are called long interspersed nuclear elements (LINEs) in vertebrates, contribute to genome structure and evolution through their replication process (5,8,10,21). In humans, LINEs have accumulated up to 21% of the genome, and they are the major source of insertional mutagenesis. LINEs shape the mammalian genome through exon shuffling, mobilization of short interspersed nuclear elements, and processed pseudogene formation (4,6,10,18,20). However, we have little knowledge of the molecular basis underlying these genomic events because the retrotransposition mechanisms of non-LTR retrotransposons are insufficiently understood, compared with those of other retroelements, such as LTR retrotransposons and retroviruses.Non-LTR retrotransposons encode an RT domain and an endonuclease (EN) domain. Pioneering studies of the Bombyx non-LTR element R2 showed that the EN domain nicks the bottom strand of target DNA and that the RT domain uses the 3Ј-hydroxyl end of the nicked DNA as a primer for reverse transcribing non-LTR element RNA (12). This reverse transcription initiation process is termed target-primed reverse transcription (TPRT) and is inherent to all non-LTR retrotransposons. During TPRT, reverse transcription is initiated at the 3Ј end of non-LTR elements. Most genomic copies of non-LTR retrotransposons are 5Ј truncated, presumably because of the arrest of reverse ...

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 TFIIIC␤ 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 TFIIIC␤. 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 boxbinding 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. RNA polymerase (pol)1 III is a multisubunit enzyme that is directed to initiate RNA synthesis by transcription factors (TFs) that bind to gene promoter elements. Pol III transcripts comprise a large variety of small nuclear and cytoplasmic RNAs (1). While there is substantial diversity in the promoter structures of pol III-transcribed genes, three major classes are responsible for the synthesis of the more abundant of the cellular pol III transcripts, tRNAs, 5 S rRNA, and U6 snRNA (2). Each of these represent one of three distinct gene classes that utilize a characteristic promoter structure and a specific set of TFs (3). 5 S rRNA genes comprise class I and contain a principal internal promoter that is a binding site for TFIIIA. Class 3 genes utilize upstream TATA elements and, in metazoans, an additional upstream element that binds a distinct multisubunit TF (4). Class 2 is represented by tRNA genes, which are driven by an internal split promoter composed of proximal box A and distal box B elements. In contrast to the diversity of promoter structures, the termination signal for polymerase III transcription is the run of dT residues found at the 3Ј-ends of pol III-transcribed genes (reviewed in Ref. 5).The A box usually begins 10 -15 base pairs (bp) downstream of the start site of transcription, and the B box is further downstream, the distance depending on the particular tRNA gene. The terminator is the most 3Ј element, usually found within 20 bp of the B box (6). The multisubunit TF IIIC spans the length of tRNA genes, binding to the internal promoter and terminator regions (3). The largest subunit of TFIIIC plays a central role in initiation by recognizing the B box promoter and orienting its associated subunits along the DNA. Th...

Our group demonstrated recently that the 3' ends of several families of tRNA-derived SINEs (short interspersed repetitive elements) originated from the 3' ends of LINEs (long interspersed repetitive elements) [Ohshima et al. (1996) Mol. Cell. Biol. 16:3756-3764]. Two fully characterized examples of such organization were provided by the tortoise Pol III/SINE and the salmonid HpaI family of SINEs, and two probable examples were provided by the tobacco TS family of SINEs and the salmon SmaI family of SINEs. This organization of SINEs can explain their potential to retropose in the genome since it appears reasonable that the sites for recognition of LINEs by reverse transcriptase should be located within the 3'-end sequences of LINEs. We now add another example to this category of SINEs. In the bovine genome, there are Bov-tA SINEs, which belong to the superfamily of tRNA-derived families of SINEs, and Bov-B LINEs, which were recently demonstrated to belong to a LINE family. Moreover, Bov-tA and Bov-B share the same 3'-end tail. We propose a possible scenario whereby the composite structure of the bovine Bov-tA family of SINEs might have been generated from the Bov-B family of LINEs during evolution.

R1Bm is a non-LTR retrotransposon found specifically within 28S rRNA genes of the silkworm. Different from other non-LTR retrotransposons encoding two open reading frames (ORFs), R1Bm structurally lacks a poly (A) tract at its 3′ end. To study how R1Bm initiates reverse transcription from the poly (A)-less template RNA, we established an in vivo retrotransposition system using recombinant baculovirus, and characterized retrotransposition activities of R1Bm. Target-primed reverse transcription (TPRT) of R1Bm occurred from the cleavage site generated by endonuclease (EN). The 147 bp of 3′-untranslated region (3′UTR) was essential for efficient retrotransposition of R1Bm. Even using the complete R1Bm element, however, reverse transcription started from various sites of the template RNA mostly with 5′-UG-3′ or 5′-UGU-3′ at their 3′ ends, which are presumably base-paired with 3′ end of the EN-digested 28S rDNA target sequence, 5′-AGTAGATAGGGACA-3′. When the downstream sequence of 28S rDNA target was added to the 3′ end of R1 unit, reverse transcription started exactly from the 3′ end of 3′UTR and retrotransposition efficiency increased. These results indicate that 3′-terminal structure of template RNA including read-through region interacts with its target rDNA sequences of R1Bm, which plays important roles in initial process of TPRT in vivo.