scholarly journals Accurate and efficient 3' processing of U2 small nuclear RNA precursor in a fractionated cytoplasmic extract.

1987 ◽  
Vol 7 (9) ◽  
pp. 3131-3137 ◽  
Author(s):  
A M Kleinschmidt ◽  
T Pederson
Keyword(s):  
Rna Polymerase Ii ◽  
Small Nuclear Rna ◽  
Rnase Protection ◽  
In Vitro Processing ◽  
S100 Extract ◽  
Processing Activity ◽  
Precursor Molecules ◽  
Trna Precursor ◽  
Rna Precursor

The small nuclear RNAs U1, U2, U4, and U5 are cofactors in mRNA splicing and, like the pre-mRNAs with which they interact, are transcribed by RNA polymerase II. Also like mRNAs, mature U1 and U2 RNAs are generated by 3' processing of their primary transcripts. In this study we have investigated the in vitro processing of an SP6-transcribed human U2 RNA precursor, the 3' end of which matches that of authentic human U2 RNA precursor molecules. Although the SP6-U2 RNA precursor was efficiently processed in an ammonium sulfate-fractionated HeLa cytoplasmic S100 extract, the product RNA was unstable. Further purification of the processing activity on glycerol gradients resolved a 7S activity that nonspecifically cleaved all RNAs tested and a 15S activity that efficiently processed the 3' end of pre-U2 RNA. The 15S activity did not process the 3' end of a tRNA precursor molecule. As demonstrated by RNase protection, the processed 3' end of the SP6-U2 RNA maps to the same nucleotides as does mature HeLa U2 RNA.

Accurate and efficient 3' processing of U2 small nuclear RNA precursor in a fractionated cytoplasmic extract

1987 ◽  
Vol 7 (9) ◽  
pp. 3131-3137
Author(s):  
A M Kleinschmidt ◽  
T Pederson
Keyword(s):  
Rna Polymerase Ii ◽  
Small Nuclear Rna ◽  
Rnase Protection ◽  
In Vitro Processing ◽  
S100 Extract ◽  
Processing Activity ◽  
Precursor Molecules ◽  
Trna Precursor ◽  
Rna Precursor

The small nuclear RNAs U1, U2, U4, and U5 are cofactors in mRNA splicing and, like the pre-mRNAs with which they interact, are transcribed by RNA polymerase II. Also like mRNAs, mature U1 and U2 RNAs are generated by 3' processing of their primary transcripts. In this study we have investigated the in vitro processing of an SP6-transcribed human U2 RNA precursor, the 3' end of which matches that of authentic human U2 RNA precursor molecules. Although the SP6-U2 RNA precursor was efficiently processed in an ammonium sulfate-fractionated HeLa cytoplasmic S100 extract, the product RNA was unstable. Further purification of the processing activity on glycerol gradients resolved a 7S activity that nonspecifically cleaved all RNAs tested and a 15S activity that efficiently processed the 3' end of pre-U2 RNA. The 15S activity did not process the 3' end of a tRNA precursor molecule. As demonstrated by RNase protection, the processed 3' end of the SP6-U2 RNA maps to the same nucleotides as does mature HeLa U2 RNA.

In vitro processing of B. mori transfer RNA precursor molecules

Cell ◽  
1979 ◽  
Vol 17 (2) ◽  
pp. 389-397 ◽  
Author(s):  
Richard L. Garber ◽  
Sidney Altman
Keyword(s):  
Transfer Rna ◽  
In Vitro Processing ◽  
Precursor Molecules ◽  
Rna Precursor

U2.3 Precursor Small Nuclear RNA in vitro Processing Assay

BIO-PROTOCOL ◽  
2021 ◽  
Vol 11 (17) ◽  
Author(s):  
Chan Lin ◽  
Yujie Feng ◽  
Xueyan Peng ◽  
Jiaming Wu ◽  
Weili Wang ◽  
...  
Keyword(s):  
Small Nuclear Rna ◽  
In Vitro Processing ◽  
Nuclear Rna

In vitro transcription of a Drosophila U1 small nuclear RNA gene requires TATA box-binding protein and two proximal cis-acting elements with stringent spacing requirements

1993 ◽  
Vol 13 (9) ◽  
pp. 5918-5927
Author(s):  
Z Zamrod ◽  
C M Tyree ◽  
Y Song ◽  
W E Stumph
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Tata Box ◽  
Start Site ◽  
Small Nuclear Rna ◽  
Wild Type ◽  
Nuclear Rna ◽  
Tata Sequence ◽  
Tata Box Binding Protein

Transcription of a Drosophila U1 small nuclear RNA gene was functionally analyzed in cell extracts derived from 0- to 12-h embryos. Two promoter elements essential for efficient initiation of transcription in vitro by RNA polymerase II were identified. The first, termed PSEA, is located between positions -41 and -61 relative to the transcription start site, is crucial for promoter activity, and is the dominant element for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vertebrate small nuclear RNA genes. The second element, termed PSEB, is located at positions -25 to -32 and is required for an efficient level of transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduced transcriptional activity relative to that of the wild-type promoter. Although the PSEB sequence does not have any obvious sequence similarity to a TATA box, conversion of PSEB to the canonical TATA sequence dramatically increased the efficiency of the U1 promoter and simultaneously relieved the requirement for the upstream PSEA. Despite these effects, introduction of the TATA sequence into the U1 promoter had no effect on the choice of start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is required for transcription from the wild-type U1 promoter as well as from the TATA-containing U1 promoter.

Human genes for U2 small nuclear RNA are tandemly repeated

1984 ◽  
Vol 4 (3) ◽  
pp. 492-499
Author(s):  
S W Van Arsdell ◽  
A M Weiner
Keyword(s):  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Initiation Site ◽  
Tandem Array ◽  
Small Nuclear Rna ◽  
Coding Region ◽  
Region I ◽  
Nuclear Rna ◽  
Region Ii

We found that the genes for human U2 small nuclear RNA (snRNA) are organized as a nearly perfect tandem array of 10 to 20 copies per haploid genome. Although the coding region for the mature form of U2 RNA was only 188 base pairs (bp) long, the basic repeating unit of the tandem array was 6 kilobase pairs in length. Comparison of DNA sequences immediately upstream from human U1 and U2 genes revealed two regions of strong homology: region I (15 bp long) lay upstream of region II (20 bp long) and was separated from it by about the same distance in U1 genes (25 bp) as in U2 genes (21 bp); however, region I and region II were located 174 bp further upstream from the 5' end of the snRNA coding sequence in U1 genes than in U2 genes. Homologs of region II were also found upstream of the snRNA coding region in a mouse U2 gene and two rat U1 genes. Murphy et al. (Cell 29:265-274, 1982) have found that sequences within region II may function as the equivalent of a TATA box for initiation by RNA polymerase II in vitro at a position 183 bp upstream from the 5' end of the human U1 snRNA coding region. In light of the data reported here, this result suggests that region II does indeed play a role in transcription but that its position relative to the actual initiation site can vary.

scholarly journals 3' processing of human pre-U2 small nuclear RNA: a base-pairing interaction between the 3' extension of the precursor and an internal region.

1997 ◽  
Vol 17 (12) ◽  
pp. 7178-7185 ◽  
Author(s):  
Q Huang ◽  
M R Jacobson ◽  
T Pederson
Keyword(s):  
Rna Polymerase Ii ◽  
Small Nuclear Rna ◽  
Wild Type ◽  
Base Pairing ◽  
Pairing Interaction ◽  
Stem Loop ◽  
Internal Region ◽  
Base Pairing Interaction

The spliceosomal small nuclear RNAs U1, U2, U4, and U5 are transcribed by RNA polymerase II as precursors with extensions at their 3' ends. The 3' processing of these pre-snRNAs is not understood in detail. Two pathways of pre-U2 RNA 3' processing in vitro were revealed in the present investigation by using a series of human wild-type and mutant pre-U2 RNAs. Substrates with wild-type 3' ends were initially shortened by three or four nucleotides (which is the first step in vivo), and the correct mature 3' end was then rapidly generated. In contrast, certain mutant pre-U2 RNAs displayed an aberrant 3' processing pathway typified by the persistence of intermediates representing cleavage at each internucleoside bond in the precursor 3' extension. Comparison of the wild-type and mutant pre-U2 RNAs revealed a potential base-pairing interaction between nucleotides in the precursor 3' extension and a sequence located between the Sm domain and stem-loop III of U2 RNA. Substrate processing competition experiments using a highly purified pre-U2 RNA 3' processing activity suggested that only RNAs capable of this base-pairing interaction had high affinity for the pre-U2 RNA 3' processing enzyme. The importance of this postulated base-pairing interaction between the precursor 3' extension and the internal region between the Sm domain and stem-loop III was confirmed by the results obtained with a compensatory substitution that restores the base pairing, which displayed the normal 3' processing reaction. These results implicate a precursor-specific base-paired structure involving sequences on both sides of the mature cleavage site in the 3' processing of human U2 RNA.

scholarly journals Composition and processing activity of a semi-recombinant holo U7 snRNP

2019 ◽  
Vol 48 (3) ◽  
pp. 1508-1530 ◽  
Author(s):  
Katarzyna Bucholc ◽  
Wei Shen Aik ◽  
Xiao-cui Yang ◽  
Kaituo Wang ◽  
Z Hong Zhou ◽  
...  
Keyword(s):  
Animal Cells ◽  
Sm Proteins ◽  
Nuclear Extracts ◽  
In Vitro Processing ◽  
Functionally Impaired ◽  
Catalytically Active ◽  
U7 Snrnp ◽  
Core Components ◽  
Processing Activity

Abstract In animal cells, replication-dependent histone pre-mRNAs are cleaved at the 3′ end by U7 snRNP consisting of two core components: a ∼60-nucleotide U7 snRNA and a ring of seven proteins, with Lsm10 and Lsm11 replacing the spliceosomal SmD1 and SmD2. Lsm11 interacts with FLASH and together they recruit the endonuclease CPSF73 and other polyadenylation factors, forming catalytically active holo U7 snRNP. Here, we assembled core U7 snRNP bound to FLASH from recombinant components and analyzed its appearance by electron microscopy and ability to support histone pre-mRNA processing in the presence of polyadenylation factors from nuclear extracts. We demonstrate that semi-recombinant holo U7 snRNP reconstituted in this manner has the same composition and functional properties as endogenous U7 snRNP, and accurately cleaves histone pre-mRNAs in a reconstituted in vitro processing reaction. We also demonstrate that the U7-specific Sm ring assembles efficiently in vitro on a spliceosomal Sm site but the engineered U7 snRNP is functionally impaired. This approach offers a unique opportunity to study the importance of various regions in the Sm proteins and U7 snRNA in 3′ end processing of histone pre-mRNAs.

scholarly journals In vitro transcription of a Drosophila U1 small nuclear RNA gene requires TATA box-binding protein and two proximal cis-acting elements with stringent spacing requirements.

1993 ◽  
Vol 13 (9) ◽  
pp. 5918-5927 ◽  
Author(s):  
Z Zamrod ◽  
C M Tyree ◽  
Y Song ◽  
W E Stumph
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Tata Box ◽  
Start Site ◽  
Small Nuclear Rna ◽  
Wild Type ◽  
Nuclear Rna ◽  
Tata Sequence ◽  
Tata Box Binding Protein

Transcription of a Drosophila U1 small nuclear RNA gene was functionally analyzed in cell extracts derived from 0- to 12-h embryos. Two promoter elements essential for efficient initiation of transcription in vitro by RNA polymerase II were identified. The first, termed PSEA, is located between positions -41 and -61 relative to the transcription start site, is crucial for promoter activity, and is the dominant element for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vertebrate small nuclear RNA genes. The second element, termed PSEB, is located at positions -25 to -32 and is required for an efficient level of transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduced transcriptional activity relative to that of the wild-type promoter. Although the PSEB sequence does not have any obvious sequence similarity to a TATA box, conversion of PSEB to the canonical TATA sequence dramatically increased the efficiency of the U1 promoter and simultaneously relieved the requirement for the upstream PSEA. Despite these effects, introduction of the TATA sequence into the U1 promoter had no effect on the choice of start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is required for transcription from the wild-type U1 promoter as well as from the TATA-containing U1 promoter.

scholarly journals Human genes for U2 small nuclear RNA are tandemly repeated.

1984 ◽  
Vol 4 (3) ◽  
pp. 492-499 ◽  
Author(s):  
S W Van Arsdell ◽  
A M Weiner
Keyword(s):  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Initiation Site ◽  
Tandem Array ◽  
Small Nuclear Rna ◽  
Coding Region ◽  
Region I ◽  
Nuclear Rna ◽  
Region Ii

We found that the genes for human U2 small nuclear RNA (snRNA) are organized as a nearly perfect tandem array of 10 to 20 copies per haploid genome. Although the coding region for the mature form of U2 RNA was only 188 base pairs (bp) long, the basic repeating unit of the tandem array was 6 kilobase pairs in length. Comparison of DNA sequences immediately upstream from human U1 and U2 genes revealed two regions of strong homology: region I (15 bp long) lay upstream of region II (20 bp long) and was separated from it by about the same distance in U1 genes (25 bp) as in U2 genes (21 bp); however, region I and region II were located 174 bp further upstream from the 5' end of the snRNA coding sequence in U1 genes than in U2 genes. Homologs of region II were also found upstream of the snRNA coding region in a mouse U2 gene and two rat U1 genes. Murphy et al. (Cell 29:265-274, 1982) have found that sequences within region II may function as the equivalent of a TATA box for initiation by RNA polymerase II in vitro at a position 183 bp upstream from the 5' end of the human U1 snRNA coding region. In light of the data reported here, this result suggests that region II does indeed play a role in transcription but that its position relative to the actual initiation site can vary.

scholarly journals A Divergent Transcription Factor TFIIB in Trypanosomes Is Required for RNA Polymerase II-Dependent Spliced Leader RNA Transcription and Cell Viability

2006 ◽  
Vol 5 (2) ◽  
pp. 293-300 ◽  
Author(s):  
Jennifer B. Palenchar ◽  
Wenzhe Liu ◽  
Peter M. Palenchar ◽  
Vivian Bellofatto
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gene Transcription ◽  
Binding Protein ◽  
In Vitro Transcription ◽  
Tata Binding Protein ◽  
Small Nuclear Rna ◽  
Spliced Leader

ABSTRACT Transcription by RNA polymerase II in trypanosomes deviates from the standard eukaryotic paradigm. Genes are transcribed polycistronically and subsequently cleaved into functional mRNAs, requiring trans splicing of a capped 39-nucleotide leader RNA derived from a short transcript, the spliced leader (SL) RNA. The only identified trypanosome RNA polymerase II promoter is that of the SL RNA gene. We have previously shown that transcription of SL RNA requires divergent trypanosome homologs of RNA polymerase II, TATA binding protein, and the small nuclear RNA (snRNA)-activating protein complex. In other eukaryotes, TFIIB is an additional key component of transcription for both mRNAs and polymerase II-dependent snRNAs. We have identified a divergent homolog of the usually highly conserved basal transcription factor, TFIIB, from the pathogenic parasite Trypanosoma brucei. T. brucei TFIIB (TbTFIIB) interacted directly with the trypanosome TATA binding protein and RNA polymerase II, confirming its identity. Functionally, in vitro transcription studies demonstrated that TbTFIIB is indispensable in SL RNA gene transcription. RNA interference (RNAi) studies corroborated the essential nature of TbTFIIB, as depletion of this protein led to growth arrest of parasites. Furthermore, nuclear extracts prepared from parasites depleted of TbTFIIB, after the induction of RNAi, required recombinant TbTFIIB to support spliced leader transcription. The information gleaned from TbTFIIB studies furthers our understanding of SL RNA gene transcription and the elusive overall transcriptional processes in trypanosomes.

Sign in / Sign up

Export Citation Format

Share Document