scholarly journals R2 Retrotransposons Encode a Self-Cleaving Ribozyme for Processing from an rRNA Cotranscript

2010 ◽  
Vol 30 (13) ◽  
pp. 3142-3150 ◽  
Author(s):  
Danna G. Eickbush ◽  
Thomas H. Eickbush
Keyword(s):  
Hepatitis Delta Virus ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Sequence Evolution ◽  
28S Rrna ◽  
Common Descent ◽  
Hdv Ribozyme ◽  
Delta Virus

ABSTRACT The non-long terminal repeat (non-LTR) retrotransposon R2 is inserted into the 28S rRNA genes of many animals. Expression of the element appears to be by cotranscription with the rRNA gene unit. We show here that processing of the rRNA cotranscript at the 5′ end of the R2 element in Drosophila simulans is rapid and utilizes an unexpected mechanism. Using RNA synthesized in vitro, the 5′ untranslated region of R2 was shown capable of rapid and efficient self-cleavage of the 28S-R2 cotranscript. The 5′ end generated in vitro by the R2 ribozyme was at the position identical to that found for in vivo R2 transcripts. The RNA segment corresponding to the R2 ribozyme could be folded into a double pseudoknot structure similar to that of the hepatitis delta virus (HDV) ribozyme. Remarkably, 21 of the nucleotide positions in and around the active site of the HDV ribozyme were identical in R2. R2 elements from other Drosophila species were also shown to encode HDV-like ribozymes capable of self-cleavage. Tracing their sequence evolution in the Drosophila lineage suggests that the extensive similarity of the R2 ribozyme from D. simulans to that of HDV was a result of convergent evolution, not common descent.

Processing of truncated mouse or human rRNA transcribed from ribosomal minigenes transfected into mouse cells

1994 ◽  
Vol 14 (6) ◽  
pp. 4044-4056
Author(s):  
K V Hadjiolova ◽  
A Normann ◽  
J Cavaillé ◽  
E Soupène ◽  
S Mazan ◽  
...  
Keyword(s):  
18S Rrna ◽  
Processing System ◽  
Rrna Genes ◽  
In Vitro Assays ◽  
Rrna Gene ◽  
28S Rrna ◽  
Rrna Processing ◽  
Mouse Cells

The processing of pre-rRNA in eukaryotic cells involves a complex pattern of nucleolytic reactions taking place in preribosomes with the participation of several nonribosomal proteins and small nuclear RNAs. The mechanism of these reactions remains largely unknown, mainly because of the absence of faithful in vitro assays for most processing steps. We have developed a pre-rRNA processing system using the transient expression of ribosomal minigenes transfected into cultured mouse cells. Truncated mouse or human rRNA genes are faithfully transcribed under the control of mouse promoter and terminator signals. The fate of these transcripts is analyzed by the use of reporter sequences flanking the rRNA gene inserts. Both mouse and human transcripts, containing the 3' end of 18S rRNA-encoding DNA (rDNA), internal transcribed spacer (ITS) 1, 5.8S rDNA, ITS 2, and the 5' end of 28S rDNA, are processed predominantly to molecules coterminal with the natural mature rRNAs plus minor products corresponding to cleavages within ITS 1 and ITS 2. To delineate cis-acting signals in pre-rRNA processing, we studied series of more truncated human-mouse minigenes. A faithful processing at the 18S rRNA/ITS 1 junction can be observed with transcripts containing only the 60 3'-terminal nucleotides of 18S rRNA and the 533 proximal nucleotides of ITS 1. However, further truncation of 18S rRNA (to 8 nucleotides) or of ITS 1 (to 48 nucleotides) abolishes the cleavage of the transcript. Processing at the ITS 2/28S rRNA junction is observed with truncated transcripts lacking the 5.8S rRNA plus a major part of ITS 2 and containing only 502 nucleotides of 28S rRNA. However, further truncation of the 28S rRNA segment to 217 nucleotides abolishes processing. Minigene transcripts containing most internal sequences of either ITS 1 or ITS 2, but devoid of ITS/mature rRNA junctions, are not processed, suggesting that the cleavages in vivo within either ITS segment are dependent on the presence in cis of mature rRNA sequences. These results show that the major cis signals for pre-rRNA processing at the 18S rRNA/ITS 1 or the ITS2/28S rRNA junction involve solely a limited critical length of the respective mature rRNA and adjacent spacer sequences.

scholarly journals Processing of truncated mouse or human rRNA transcribed from ribosomal minigenes transfected into mouse cells.

1994 ◽  
Vol 14 (6) ◽  
pp. 4044-4056 ◽  
Author(s):  
K V Hadjiolova ◽  
A Normann ◽  
J Cavaillé ◽  
E Soupène ◽  
S Mazan ◽  
...  
Keyword(s):  
18S Rrna ◽  
Processing System ◽  
Rrna Genes ◽  
In Vitro Assays ◽  
Rrna Gene ◽  
28S Rrna ◽  
Rrna Processing ◽  
Mouse Cells

The processing of pre-rRNA in eukaryotic cells involves a complex pattern of nucleolytic reactions taking place in preribosomes with the participation of several nonribosomal proteins and small nuclear RNAs. The mechanism of these reactions remains largely unknown, mainly because of the absence of faithful in vitro assays for most processing steps. We have developed a pre-rRNA processing system using the transient expression of ribosomal minigenes transfected into cultured mouse cells. Truncated mouse or human rRNA genes are faithfully transcribed under the control of mouse promoter and terminator signals. The fate of these transcripts is analyzed by the use of reporter sequences flanking the rRNA gene inserts. Both mouse and human transcripts, containing the 3' end of 18S rRNA-encoding DNA (rDNA), internal transcribed spacer (ITS) 1, 5.8S rDNA, ITS 2, and the 5' end of 28S rDNA, are processed predominantly to molecules coterminal with the natural mature rRNAs plus minor products corresponding to cleavages within ITS 1 and ITS 2. To delineate cis-acting signals in pre-rRNA processing, we studied series of more truncated human-mouse minigenes. A faithful processing at the 18S rRNA/ITS 1 junction can be observed with transcripts containing only the 60 3'-terminal nucleotides of 18S rRNA and the 533 proximal nucleotides of ITS 1. However, further truncation of 18S rRNA (to 8 nucleotides) or of ITS 1 (to 48 nucleotides) abolishes the cleavage of the transcript. Processing at the ITS 2/28S rRNA junction is observed with truncated transcripts lacking the 5.8S rRNA plus a major part of ITS 2 and containing only 502 nucleotides of 28S rRNA. However, further truncation of the 28S rRNA segment to 217 nucleotides abolishes processing. Minigene transcripts containing most internal sequences of either ITS 1 or ITS 2, but devoid of ITS/mature rRNA junctions, are not processed, suggesting that the cleavages in vivo within either ITS segment are dependent on the presence in cis of mature rRNA sequences. These results show that the major cis signals for pre-rRNA processing at the 18S rRNA/ITS 1 or the ITS2/28S rRNA junction involve solely a limited critical length of the respective mature rRNA and adjacent spacer sequences.

scholarly journals Genetic relationships of Corynebacterium diphtheriae strains isolated from a diphtheria case and carriers by restriction fragment length polymorphism of rRNA genes

1995 ◽  
Vol 37 (4) ◽  
pp. 291-296
Author(s):  
Claudio Tavares Sacchi ◽  
Ana Paula Silva de Lemos ◽  
Silvana Tadeu Casagrande ◽  
Alice Massumi Mori ◽  
Carmecy Lopes de Almeida
Keyword(s):  
Cell Culture ◽  
Genetic Relationships ◽  
Culture Method ◽  
Vero Cells ◽  
Corynebacterium Diphtheriae ◽  
Rrna Genes ◽  
Diffusion Method ◽  
Rrna Gene

In the present study we report the results of an analysis, based on ribotyping of Corynebacterium diphtheriae intermedius strains isolated from a 9 years old child with clinical diphtheria and his 5 contacts. Quantitative analysis of RFLPs of rRNA was used to determine relatedness of these 7 C.diphtheriae strains providing support data in the diphtheria epidemiology. We have also tested those strains for toxigenicity in vitro by using the Elek's gel diffusion method and in vivo by using cell culture method on cultured monkey kidney cell (VERO cells). The hybridization results revealed that the 5 C.diphtheriae strains isolated from contacts and one isolated from the clinical case (nose case strain) had identical RFLP patterns with all 4 restriction endonucleases used, ribotype B. The genetic distance from this ribotype and ribotype A (throat case strain), that we initially assumed to be responsible for the illness of the patient, was of 0.450 showing poor genetic correlation among these two ribotypes. We found no significant differences concerned to the toxin production by using the cell culture method. In conclusion, the use of RFLPs of rRNA gene was successful in detecting minor differences in closely related toxigenic C.diphtheriae intermedius strains and providing information about genetic relationships among them.

scholarly journals Hepatitis delta virus facilitates the selection of hepatitis B virus mutants in vivo and functionally impacts on their replicative capacity in vitro

2016 ◽  
Vol 22 (1) ◽  
pp. 98.e1-98.e6 ◽  
Author(s):  
E. Shirvani-Dastgerdi ◽  
M.R. Pourkarim ◽  
U. Herbers ◽  
S. Amini-Bavil-Olyaee ◽  
E. Yagmur ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Hepatitis Delta Virus ◽  
Replicative Capacity ◽  
Hepatitis Delta ◽  
B Virus ◽  
Selection Of ◽  
Delta Virus

Hepatitis Delta Virus can persist and propagate through human cell division both in vitro and in vivo

2015 ◽  
Vol 53 (01) ◽  
Author(s):  
OD Bhadra ◽  
K Giersch ◽  
T Volz ◽  
L Allweiss ◽  
AW Lohse ◽  
...  
Keyword(s):  
Cell Division ◽  
Human Cell ◽  
Hepatitis Delta Virus ◽  
Hepatitis Delta ◽  
Delta Virus

Hepatitis delta virus persists during liver regeneration and is amplified through cell division both in vitro and in vivo

Gut ◽  
2017 ◽  
Vol 68 (1) ◽  
pp. 150-157 ◽  
Author(s):  
Katja Giersch ◽  
Oliver D Bhadra ◽  
Tassilo Volz ◽  
Lena Allweiss ◽  
Kristoffer Riecken ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Liver Regeneration ◽  
Hepatitis Delta Virus ◽  
Core Antigen ◽  
Hepatitis Delta ◽  
Dividing Cells ◽  
Humanised Mice ◽  
Delta Virus

ObjectiveHepatitis delta virus (HDV) was shown to persist for weeks in the absence of HBV and for months after liver transplantation, demonstrating the ability of HDV to persevere in quiescent hepatocytes. The aim of the study was to evaluate the impact of cell proliferation on HDV persistence in vitro and in vivo.DesignGenetically labelled human sodium taurocholate cotransporting polypeptide (hNTCP)-transduced human hepatoma(HepG2) cells were infected with HBV/HDV and passaged every 7 days for 100 days in the presence of the entry inhibitor Myrcludex-B. In vivo, cell proliferation was triggered by transplanting primary human hepatocytes (PHHs) isolated from HBV/HDV-infected humanised mice into naïve recipients. Virological parameters were measured by quantitative real time polymerase chain reaction (qRT-PCR). Hepatitis delta antigen (HDAg), hepatitis B core antigen (HBcAg) and cell proliferation were determined by immunofluorescence.ResultsDespite 15 in vitro cell passages and block of viral spreading by Myrcludex-B, clonal cell expansion permitted amplification of HDV infection. In vivo, expansion of PHHs isolated from HBV/HDV-infected humanised mice was confirmed 3 days, 2, 4 and 8 weeks after transplantation. While HBV markers rapidly dropped in proliferating PHHs, HDAg-positive hepatocytes were observed among dividing cells at all time points. Notably, HDAg-positive cells appeared in clusters, indicating that HDV was transmitted to daughter cells during liver regeneration even in the absence of de novo infection.ConclusionThis study demonstrates that HDV persists during liver regeneration by transmitting HDV RNA to dividing cells even in the absence of HBV coinfection. The strong persistence capacities of HDV may also explain why HDV clearance is difficult to achieve in HBV/HDV chronically infected patients.

scholarly journals Resistance of Human Hepatitis Delta Virus RNAs to Dicer Activity

2003 ◽  
Vol 77 (22) ◽  
pp. 11910-11917 ◽  
Author(s):  
Jinhong Chang ◽  
Patrick Provost ◽  
John M. Taylor
Keyword(s):  
Hepatitis Delta Virus ◽  
Unit Length ◽  
Potato Spindle Tuber Viroid ◽  
Micro Rna ◽  
Hepatitis Delta ◽  
Human Hepatitis ◽  
Putative Precursor ◽  
Delta Virus

ABSTRACT The endonuclease dicer cleaves RNAs that are 100% double stranded and certain RNAs with extensive but <100% pairing to release ∼21-nucleotide (nt) fragments. Circular 1,679-nt genomic and antigenomic RNAs of human hepatitis delta virus (HDV) can fold into a rod-like structure with 74% pairing. However, during HDV replication in hepatocytes of human, woodchuck, and mouse origin, no ∼21-nt RNAs were detected. Likewise, in vitro, purified recombinant dicer gave <0.2% cleavage of unit-length HDV RNAs. Similarly, rod-like RNAs of potato spindle tuber viroid (PSTVd) and avocado sunblotch viroid (ASBVd) were only 0.5% cleaved. Furthermore, when a 66-nt hairpin RNA with 79% pairing, the putative precursor to miR-122, which is an abundant liver micro-RNA, replaced one end of HDV genomic RNA, it was poorly cleaved, both in vivo and in vitro. In contrast, this 66-nt hairpin, in the absence of appended HDV sequences, was >80% cleaved in vitro. Other 66-nt hairpins derived from one end of genomic HDV, PSTVd, or ASBVd RNAs were also cleaved. Apparently, for unit-length RNAs of HDV, PSTVd, and ASBVd, it is the extended structure with <100% base pairing that confers significant resistance to dicer action.

O012 : Hepatitis delta virus can survive liver regeneration and is amplified through human cell division both in vitro and in vivo

2015 ◽  
Vol 62 ◽  
pp. S195-S196 ◽  
Author(s):  
K. Giersch ◽  
O.D. Bhadra ◽  
T. Volz ◽  
L. Allweiss ◽  
A.W. Lohse ◽  
...  
Keyword(s):  
Cell Division ◽  
Liver Regeneration ◽  
Human Cell ◽  
Hepatitis Delta Virus ◽  
Hepatitis Delta ◽  
Delta Virus

scholarly journals Initiation of Hepatitis Delta Virus Genome Replication

1998 ◽  
Vol 72 (6) ◽  
pp. 4783-4788 ◽  
Author(s):  
Kate Dingle ◽  
Vadim Bichko ◽  
Harmon Zuccola ◽  
James Hogle ◽  
John Taylor
Keyword(s):  
Hepatitis Delta Virus ◽  
Rna Synthesis ◽  
De Novo ◽  
Virus Genome ◽  
Northern Analysis ◽  
Genome Replication ◽  
Hepatitis Delta ◽  
Delta Virus

ABSTRACT The small, 195-amino-acid form of the hepatitis delta virus (HDV) antigen (δAg-S) is essential for genome replication, i.e., for the transcription, processing, and accumulation of HDV RNAs. To better understand this requirement, we used purified recombinant δAg-S and HDV RNA synthesized in vitro to assemble high-molecular-weight ribonucleoprotein (RNP) structures. After transfection of these RNPs into human cells, we detected HDV genome replication, as assayed by Northern analysis or immunofluorescence microscopy. Our interpretation is that the input δAg-S is necessary for the RNA to undergo limited amounts of RNA-directed RNA synthesis, RNA processing, and mRNA formation, leading to de novo translation of δAg-S. It is this second source of δAg-S which then goes on to support genome replication. This assay made it possible to manipulate in vitro the composition of the RNP and then test in vivo the ability of the complex to initiate RNA-directed RNA synthesis and go on to achieve genome replication. For example, both genomic and antigenomic linear RNAs were acceptable. Substitution for δAg-S with truncated or modified forms of the δAg, and even with HIV nucleocapsid protein and polylysine, was unacceptable; the exception was a form of δAg-S with six histidines added at the C terminus. We expect that further in vitro modifications of these RNP complexes should help define the in vivo requirements for what we define as the initiation of HDV genome replication.

scholarly journals Replication of the Human Hepatitis Delta Virus Genome Is Initiated in Mouse Hepatocytes following Intravenous Injection of Naked DNA or RNA Sequences

2001 ◽  
Vol 75 (7) ◽  
pp. 3469-3473 ◽  
Author(s):  
Jinhong Chang ◽  
Luis J. Sigal ◽  
Anthony Lerro ◽  
John Taylor
Keyword(s):  
Hepatitis Delta Virus ◽  
Virus Genome ◽  
In Vivo Studies ◽  
Genome Replication ◽  
Rna Sequences ◽  
Hepatitis Delta ◽  
Naked Dna ◽  
Delta Virus

ABSTRACT As early as 5 days after DNA copies of the hepatitis delta virus (HDV) genome or even in vitro-transcribed HDV RNA sequences were injected into the mouse tail vein using the hydrodynamics-based transfection procedure of F. Liu et al. (Gene Ther. 6:1258–1266, 1999), it was possible to detect in the liver by Northern analyses of RNA, immunoblots of protein, and immunostaining of liver sections what were considered typical features of HDV genome replication. This transfection strategy should have valuable applications for in vivo studies of HDV replication and pathogenesis and may also be useful for studies of other hepatotropic viruses.

Sign in / Sign up

Export Citation Format

Share Document