Mapping the 5′ and the 3′ ends of<i>Tetrahymena thermophila</i>mRNAs using RNA ligase mediated amplification of cDNA ends (RLM-RACE)

Liu, X.; Gorovsky, M.A.

doi:10.1093/nar/22.3.551

Cited by 28 publications

(36 citation statements)

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“…Considering that miR173, an initiator of TAS1 and TAS2 tasiRNA, was decreased to some degree in TGBp1 transformants while miR390, an initiator of TAS3 tasiRNA, was not affected ( Figure 1C), it is still possible that the inhibition of tasiRNA synthesis by TGBp1 is caused by the reduced efficiency of primary cleavage of the TAS transcript. Thus, we performed RNA ligase-mediated 59 rapid amplification of cDNA ends (RLM-59 RACE) PCR (Liu and Gorovsky, 1993) to detect the 39 cleavage products of the primary TAS transcripts. In wild-type Col-0 and GUS transformants, primary cleavage products of TAS2 and TAS3 precursors guided by miR173 and miR390, 313 and 77 bp, respectively, were nearly undetectable (Figures 2A and 2B, lanes 1 and 2).…”

Section: Tgbp1 Does Not Affect Mirna-directed Cleavage Of Tas Transcrmentioning

confidence: 99%

In Planta Recognition of a Double-Stranded RNA Synthesis Protein Complex by a Potexviral RNA Silencing Suppressor

et al. 2014

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RNA silencing plays an important antiviral role in plants and invertebrates. To counteract antiviral RNA silencing, most plant viruses have evolved viral suppressors of RNA silencing (VSRs). TRIPLE GENE BLOCK PROTEIN1 (TGBp1) of potexviruses is a well-characterized VSR, but the detailed mechanism by which it suppresses RNA silencing remains unclear. We demonstrate that transgenic expression of TGBp1 of plantago asiatica mosaic virus (PlAMV) induced developmental abnormalities in Arabidopsis thaliana similar to those observed in mutants of SUPPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6) required for the trans-acting small interfering RNA synthesis pathway. PlAMV-TGBp1 inhibits SGS3/ RDR6-dependent double-stranded RNA synthesis in the trans-acting small interfering RNA pathway. TGBp1 interacts with SGS3 and RDR6 and coaggregates with SGS3/RDR6 bodies, which are normally dispersed in the cytoplasm. In addition, TGBp1 forms homooligomers, whose formation coincides with TGBp1 aggregation with SGS3/RDR6 bodies. These results reveal the detailed molecular function of TGBp1 as a VSR and shed new light on the SGS3/RDR6-dependent double-stranded RNA synthesis pathway as another general target of VSRs.

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Section: Tgbp1 Does Not Affect Mirna-directed Cleavage Of Tas Transcrmentioning

confidence: 99%

In Planta Recognition of a Double-Stranded RNA Synthesis Protein Complex by a Potexviral RNA Silencing Suppressor

et al. 2014

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“…The 5Ј ends of transcripts were amplified by ligation of an RNA adapter, followed by RT-PCR (59). The method was carried out with the aid of a kit (FirstChoice RLM-RACE, product 1700; Ambion).…”

Section: Ltr-ltr Amplificationmentioning

confidence: 99%

Cassandraretrotransposons carry independently transcribed 5S RNA

Kalendar

Tanskanen

Chang

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

125

148

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We report a group of TRIMs (terminal-repeat retrotransposons in miniature), which are small nonautonomous retrotransposons. These elements, named Cassandra, universally carry conserved 5S RNA sequences and associated RNA polymerase (pol) III promoters and terminators in their long terminal repeats (LTRs). They were found in all vascular plants investigated. Uniquely for LTR retrotransposons, Cassandra produces noncapped, polyadenylated transcripts from the 5S pol III promoter. Capped, read-through transcripts containing Cassandra sequences can also be detected in RNA and in EST databases. The predicted Cassandra RNA 5S secondary structures resemble those for cellular 5S rRNA, with high information content specifically in the pol III promoter region. Genic integration sites are common for Cassandra, an unusual feature for abundant retrotransposons. The 5S in each LTR produces a tandem 5S arrangement with an inter-5S spacing resembling that of cellular 5S. The distribution of 5S genes is very variable in flowering plants and may be partially explained by Cassandra activity. Cassandra thus appears both to have adapted a ubiquitous cellular gene for ribosomal RNA for use as a promoter and to parasitize an as-yet-unidentified group of retrotransposons for the proteins needed in its lifecycle.pol III ͉ genome evolution ͉ transcription ͉ transposable element R etrotransposons, excepting SINEs (short interspersed nuclear elements) and LINEs (long interspersed nuclear elements), resemble retroviruses in their structure and intracellular life cycle. They are ubiquitous in the genomes of plants, animals, and fungi and account for Ͼ50% of large plant genomes (1, 2). Their life cycle comprises transcription of genomic copies, translation of their encoded proteins, packaging of the transcripts into virus-like particles, reverse transcription, and targeting of the cDNA copy to the nucleus for integration into the genome (3, 4). The transcriptional signals for RNA polymerase II (pol II) are found in the long terminal repeats (LTRs) at either end of the element, flanking the priming sites for reverse transcription and the coding domain specifying the proteins needed for replication and integration [supporting information (SI) Fig. S1].In addition to the classical retrotransposons, several well conserved nonautonomous groups have been discovered that lack all or part of their coding capacity (5). The BARE2 elements cannot express the capsid protein GAG (6), and Morgane lacks most of its coding capacity (7). The TRIM (terminal repeat retrotransposon in miniature) and LARD (large retrotransposon derivative) elements (Fig. S1) entirely lack reading frames for retrotransposon proteins (8-12). The TRIM elements are composed of 100-to 250-bp LTRs, priming sites for reverse transcriptase, and a small intervening segment. Evidence for past mobility suggests that they are activated by transcomplementation (10). These have been found in at least 13 species from four plant families (9, 10).Here, we describe a group of TRIM elements, which w...

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“…PCR products were separated on 2% or 3% agarose minigels and in some cases analysed by southern blotting. The D. melanogaster histone H3.3A probe was as described by Akhmanova et al, 1995. To compare non-polyadenylated and polyadenylated 3' ends, RNA-ligase-mediated rapid amplification of cDNA ends (RLM-RACE; Liu and Gorovsky, 1993) was used. Oligonucleotide 1 with the sequence 5'-TTTCTAGACTGGCCGTCGTTTTACA was ligated to the 3' ends of total RNA as described before by Liu and Gorovsky, 1993.…”

Section: Determination Of Polyadenylation Sites By Rt-pcrmentioning

confidence: 99%

“…The D. melanogaster histone H3.3A probe was as described by Akhmanova et al, 1995. To compare non-polyadenylated and polyadenylated 3' ends, RNA-ligase-mediated rapid amplification of cDNA ends (RLM-RACE; Liu and Gorovsky, 1993) was used. Oligonucleotide 1 with the sequence 5'-TTTCTAGACTGGCCGTCGTTTTACA was ligated to the 3' ends of total RNA as described before by Liu and Gorovsky, 1993. The cDNA first strand was subsequently synthesized in the presence of oligonucleotide 2 with the sequence 5'-TGTAAAACGACGGCCAGT, which was complementary to the 3' part of the oligonucleotide 1.…”

Section: Determination Of Polyadenylation Sites By Rt-pcrmentioning

confidence: 99%

“…As the next step, we analysed the 3' ends of the non-polyadenylated histone mRNAs, using the RNA-ligasemediated rapid amplification of the cDNA ends (RLM-RACE) techique (Liu and Gorovsky, 1993). An oligonucleotide (oligonucleotide 1, see Materials and Methods section) was ligated to the 3' ends of the RNA, and this RNA was used as a template for cDNA synthesis in the presence of oligonucleotide 2, which was complementary to the 3' part of oligonucleotide 1.…”

Section: T T G C a G A T A A A G C G C T A G C G T A C T C T A T M mentioning

confidence: 99%

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Two Types of Polyadenated mRNAs are Synthesized from Drosophila Replication‐Dependent Histone Genes

Akhmanova¹,

Miedema²,

Kremer³

et al. 1997

European Journal of Biochemistry

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The polyadenylation of replication-dependent histone H2B, H 3 and H4 mRNAs in Drosophilu melanoguster was analysed. Two types of mRNAs, containing a poly(A) tail, can be detected in addition to non-polyadenylated messengers, which represent the majority of replication-dependent histone mRNAs. Firstly, conventional polyadenylation signals, localized downstream from the stem-loop region, are used to produce polyadenylated mRNAs. The messengers of this type, generated from the D. melanogaster H2B gene, are preferentially synthesized in the testis of the fly. Secondly, a distinct type of polyadenylated histone mRNA has been identified. This mRNA, which is present in many different tissues and constitutes a minor part of the total histone mRNA pool, contains a short poly(A) tail, added to the end of the 3' terminal stem-loop structure, which is in most cases lacking several nucleotides from its 3' end. The sites of polyadenylation within the stem-loop are not preceded by a nornial polyadenylation signal. The possible functions of the polyadenylated histone transcripts are discussed.

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Mapping the 5′ and the 3′ ends ofTetrahymena thermophilamRNAs using RNA ligase mediated amplification of cDNA ends (RLM-RACE)

Cited by 28 publications

References 0 publications

In Planta Recognition of a Double-Stranded RNA Synthesis Protein Complex by a Potexviral RNA Silencing Suppressor

In Planta Recognition of a Double-Stranded RNA Synthesis Protein Complex by a Potexviral RNA Silencing Suppressor

Cassandraretrotransposons carry independently transcribed 5S RNA

Two Types of Polyadenated mRNAs are Synthesized from Drosophila Replication‐Dependent Histone Genes

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