Mitsuhiro Hamada

The 3′ ends of tRNA-derived SINEs originated from the 3’ ends of LINEs: A new example from the bovine genome

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 Hpal family of SINEs, and two probable examples were provided by the tobacco TS family of SINEs and the salmon Smal 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.

Essential motifs in the 3' untranslated region required for retrotransposition and the precise start of reverse transcription in non-long-terminal-repeat retrotransposon SART1.

ABSTRACT 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.

Functional roles of 3′-terminal structures of template RNA during in vivo retrotransposition of non-LTR retrotransposon, R1Bm

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.

Essential Domains for Ribonucleoprotein Complex Formation Required for Retrotransposition of Telomere-Specific Non-Long Terminal Repeat Retrotransposon SART1

ABSTRACT Non-long terminal repeat (LTR) retrotransposons are major components of the higher eukaryotic genome. Most of them have two open reading frames (ORFs): ORF2 encodes mainly the endonuclease and reverse transcriptase domains, but the functional features of ORF1 remain largely unknown. We used telomere-specific non-LTR retrotransposon SART1 in Bombyx mori and clarified essential roles of the ORF1 protein (ORF1p) in ribonucleoprotein (RNP) formation by novel approaches: in vitro reconstitution and in vivo/in vitro retrotransposition assays using the baculovirus expression system. Detailed mutation analyses showed that each of the three CCHC motifs at the ORF1 C terminus are essential for SART1 retrotransposition and are involved in packaging the SART1 mRNA specifically into RNP. We also demonstrated that amino acid residues 555 to 567 and 285 to 567 in the SART1 ORF1p are crucial for the ORF1p-ORF1p and ORF1p-ORF2p interactions, respectively. The loss of these domains abolishes protein-protein interaction, leading to SART1 retrotransposition deficiency. These data suggest that systematic formation of RNP composed of ORF1p, ORF2p, and mRNA is mainly mediated by ORF1p domains and is a common, essential step for many non-LTR retrotransposons encoding the two ORFs.

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. Copyright

RNA Polymerase III from the Fission Yeast, Schizosaccharomyces pombe

Publisher Summary This chapter reviews the RNA polymerase III (Pol III) which is a nuclear enzyme that has been specialized to produce small nontranslated RNAs in great abundance. Observation reveal that the Pol III system of the fission yeast Schizosaccharomyces pombe differs from the well-characterized S. cerevisiae, and the human Pol III systems requires a precisely positioned upstream TATA elements for function. These observations, together with observed differences among different model eukaryotes, in Pol III termination indicate that S. pombe provides novel insights in transcription mechanisms. The chapter also describes the immunoaffinity purification of epitope-tagged Pol III from S. pombe, the use of TspRI-generated templates that contain a 9 nucleotide 30 overhang for use in promoter-independent transcription, and other methods for functional analyses in vitro.