A uridylate tract mediates efficient heterogeneous nuclear ribonucleoprotein C protein-RNA cross-linking and functionally substitutes for the downstream element of the polyadenylation signal

1990 ◽  
Vol 10 (12) ◽  
pp. 6397-6407
Author(s):  
J Wilusz ◽  
T Shenk
Keyword(s):  
Spatial Relationship ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Cross Linking ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Hnrnp C ◽  
C Proteins ◽  
Nuclear Ribonucleoprotein

Every RNA added to an in vitro polyadenylation extract became stably associated with both the heterogeneous nuclear ribonucleoprotein (hnRNP) A and C proteins, as assayed by immunoprecipitation analysis using specific monoclonal antibodies. UV-cross-linking analysis, however, which assays the specific spatial relationship of certain amino acids and RNA bases, indicated that the hnRNP C proteins, but not the A proteins, were associated with downstream sequences of the simian virus 40 late polyadenylation signal in a sequence-mediated manner. A tract of five consecutive uridylate residues was required for this interaction. The insertion of a five-base U tract into a pGEM4 polylinker-derived transcript was sufficient to direct sequence-specific cross-linking of the C proteins to RNA. Finally, the five-base uridylate tract restored efficient in vitro processing to several independent poly(A) signals in which it substituted for downstream element sequences. The role of the downstream element in polyadenylation efficiency, therefore, may be mediated by sequence-directed alignment or phasing of an hnRNP complex.

scholarly journals A uridylate tract mediates efficient heterogeneous nuclear ribonucleoprotein C protein-RNA cross-linking and functionally substitutes for the downstream element of the polyadenylation signal.

1990 ◽  
Vol 10 (12) ◽  
pp. 6397-6407 ◽  
Author(s):  
J Wilusz ◽  
T Shenk
Keyword(s):  
Spatial Relationship ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Cross Linking ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Hnrnp C ◽  
C Proteins ◽  
Nuclear Ribonucleoprotein

Every RNA added to an in vitro polyadenylation extract became stably associated with both the heterogeneous nuclear ribonucleoprotein (hnRNP) A and C proteins, as assayed by immunoprecipitation analysis using specific monoclonal antibodies. UV-cross-linking analysis, however, which assays the specific spatial relationship of certain amino acids and RNA bases, indicated that the hnRNP C proteins, but not the A proteins, were associated with downstream sequences of the simian virus 40 late polyadenylation signal in a sequence-mediated manner. A tract of five consecutive uridylate residues was required for this interaction. The insertion of a five-base U tract into a pGEM4 polylinker-derived transcript was sufficient to direct sequence-specific cross-linking of the C proteins to RNA. Finally, the five-base uridylate tract restored efficient in vitro processing to several independent poly(A) signals in which it substituted for downstream element sequences. The role of the downstream element in polyadenylation efficiency, therefore, may be mediated by sequence-directed alignment or phasing of an hnRNP complex.

scholarly journals The C proteins of heterogeneous nuclear ribonucleoprotein complexes interact with RNA sequences downstream of polyadenylation cleavage sites.

1988 ◽  
Vol 8 (10) ◽  
pp. 4477-4483 ◽  
Author(s):  
J Wilusz ◽  
D I Feig ◽  
T Shenk
Keyword(s):  
Cleavage Site ◽  
Uv Light ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cross Linking ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Ribonucleoprotein Complexes ◽  
Nuclear Extracts ◽  
C Proteins ◽  
Nuclear Ribonucleoprotein

The heterogeneous nuclear ribonucleoprotein C1 and C2 proteins were preferentially cross-linked by treatment with UV light in nuclear extracts to RNAs containing six different polyadenylation signals. The domain required for the interaction was located downstream of the poly(A) cleavage site, since deletion of this segment from several polyadenylation substrate RNAs greatly reduced cross-linking efficiency. In addition, RNAs containing only downstream sequences were efficiently cross-linked to C proteins, while fully processed, polyadenylated RNAs were not. Analysis of mutated variants of the simian virus 40 late polyadenylation signal showed that uridylate-rich sequences located in the region between 30 and 55 nucleotides downstream of the cleavage site were required for efficient cross-linking of C proteins. This downstream domain of the simian virus 40 late poly(A) addition signal has been shown to influence the efficiency of the polyadenylation reaction. However, there was not a strict correlation between cross-linking of C proteins and the efficiency of polyadenylation.

The C proteins of heterogeneous nuclear ribonucleoprotein complexes interact with RNA sequences downstream of polyadenylation cleavage sites

1988 ◽  
Vol 8 (10) ◽  
pp. 4477-4483
Author(s):  
J Wilusz ◽  
D I Feig ◽  
T Shenk
Keyword(s):  
Cleavage Site ◽  
Uv Light ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cross Linking ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Ribonucleoprotein Complexes ◽  
Nuclear Extracts ◽  
C Proteins ◽  
Nuclear Ribonucleoprotein

The heterogeneous nuclear ribonucleoprotein C1 and C2 proteins were preferentially cross-linked by treatment with UV light in nuclear extracts to RNAs containing six different polyadenylation signals. The domain required for the interaction was located downstream of the poly(A) cleavage site, since deletion of this segment from several polyadenylation substrate RNAs greatly reduced cross-linking efficiency. In addition, RNAs containing only downstream sequences were efficiently cross-linked to C proteins, while fully processed, polyadenylated RNAs were not. Analysis of mutated variants of the simian virus 40 late polyadenylation signal showed that uridylate-rich sequences located in the region between 30 and 55 nucleotides downstream of the cleavage site were required for efficient cross-linking of C proteins. This downstream domain of the simian virus 40 late poly(A) addition signal has been shown to influence the efficiency of the polyadenylation reaction. However, there was not a strict correlation between cross-linking of C proteins and the efficiency of polyadenylation.

UV cross-linking of polypeptides associated with 3'-terminal exons

1990 ◽  
Vol 10 (11) ◽  
pp. 5937-5944
Author(s):  
D T Stolow ◽  
S M Berget
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Transcription Unit ◽  
Cross Linking ◽  
Consensus Sequences ◽  
Hnrnp C ◽  
Chimeric Rna ◽  
Precursor Rna ◽  
A Site

Association of nuclear proteins with chimeric vertebrate precursor RNAs containing both polyadenylation signals and an intron was examined by UV cross-linking. One major difference in cross-linking pattern was observed between this chimeric precursor RNA and precursors containing only polyadenylation or splicing signals. The heterogeneous nuclear ribonucleoprotein (hnRNP) polypeptide C cross-linked strongly to sequences downstream of the A addition site in polyadenylation precursor RNA containing only the polyadenylation signal from the simian virus 40 (SV40) late transcription unit. In contrast, the hnRNP C polypeptide cross-linked to chimeric RNA containing the same SV40 late poly(A) cassette very poorly, at a level less than 5% of that observed with the precursor RNA containing just the poly(A) site. Observation that cross-linking of the hnRNP C polypeptide to elements within the SV40 late poly(A) site was altered by the presence of an upstream intron suggests differences in the way nuclear factors associate with poly(A) sites in the presence and absence of an upstream intron. Cross-linking of C polypeptide to chimeric RNA increased with RNAs mutated for splicing or polyadenylation consensus sequences and under reaction conditions (high magnesium) that inhibited polyadenylation. Furthermore, cross-linking of hnRNP C polypeptide to precursors containing just the SV40 late poly(A) site was eliminated in the presence of competing poly(U); polyadenylation, however, was unaffected. Correlation of loss of activity with high levels of hnRNP C polypeptide cross-linking raises questions about the specificity of the interaction between the hnRNP C polypeptide and polyadenylation precursor RNAs in vitro.

scholarly journals UV cross-linking of polypeptides associated with 3'-terminal exons.

1990 ◽  
Vol 10 (11) ◽  
pp. 5937-5944 ◽  
Author(s):  
D T Stolow ◽  
S M Berget
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Transcription Unit ◽  
Cross Linking ◽  
Consensus Sequences ◽  
Hnrnp C ◽  
Chimeric Rna ◽  
Precursor Rna ◽  
A Site

Association of nuclear proteins with chimeric vertebrate precursor RNAs containing both polyadenylation signals and an intron was examined by UV cross-linking. One major difference in cross-linking pattern was observed between this chimeric precursor RNA and precursors containing only polyadenylation or splicing signals. The heterogeneous nuclear ribonucleoprotein (hnRNP) polypeptide C cross-linked strongly to sequences downstream of the A addition site in polyadenylation precursor RNA containing only the polyadenylation signal from the simian virus 40 (SV40) late transcription unit. In contrast, the hnRNP C polypeptide cross-linked to chimeric RNA containing the same SV40 late poly(A) cassette very poorly, at a level less than 5% of that observed with the precursor RNA containing just the poly(A) site. Observation that cross-linking of the hnRNP C polypeptide to elements within the SV40 late poly(A) site was altered by the presence of an upstream intron suggests differences in the way nuclear factors associate with poly(A) sites in the presence and absence of an upstream intron. Cross-linking of C polypeptide to chimeric RNA increased with RNAs mutated for splicing or polyadenylation consensus sequences and under reaction conditions (high magnesium) that inhibited polyadenylation. Furthermore, cross-linking of hnRNP C polypeptide to precursors containing just the SV40 late poly(A) site was eliminated in the presence of competing poly(U); polyadenylation, however, was unaffected. Correlation of loss of activity with high levels of hnRNP C polypeptide cross-linking raises questions about the specificity of the interaction between the hnRNP C polypeptide and polyadenylation precursor RNAs in vitro.

scholarly journals Requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation of a simian virus 40 early pre-RNA in vitro.

1987 ◽  
Vol 7 (1) ◽  
pp. 495-503 ◽  
Author(s):  
L C Ryner ◽  
J L Manley
Keyword(s):  
Simian Virus 40 ◽  
Nuclear Extract ◽  
Structural Features ◽  
Simian Virus ◽  
Micrococcal Nuclease ◽  
Small Nuclear Ribonucleoprotein ◽  
Cleavage And Polyadenylation ◽  
Nuclear Ribonucleoprotein ◽  
Cell Nuclear Extract

Using a pre-RNA containing the simian virus 40 early introns and poly(A) addition site, we investigated several possible requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation in a HeLa cell nuclear extract. Splicing and 3' end formation occurred under the same conditions but did not appear to be coupled in any way in vitro. Like splicing, 3' end cleavage and polyadenylation each required Mg2+, although spermidine could substitute in the cleavage reaction. Additionally, cleavage of this pre-RNA, but not others, was totally blocked by EDTA, indicating that structural features of pre-RNA may affect the ionic requirements of 3' end formation. The ATP analog 3' dATP inhibited both cleavage and polyadenylation even in the presence of ATP, possibly reflecting the coupled nature of these activities. A 5' cap structure appears not to be required for mRNA 3' end processing in vitro because neither the presence or absence of a 5' cap on the pre-RNA nor the addition of cap analogs to reaction mixtures had any effect on the efficiency of 3' end processing. Micrococcal nuclease pretreatment of the nuclear extract inhibited cleavage and polyadenylation. However, restoration of activity was achieved by addition of purified Escherichia coli RNA, suggesting that the inhibition caused by such a nuclease treatment was due to a general requirement for mass of RNA rather than to destruction of a particular nucleic acid-containing component such as a small nuclear ribonucleoprotein.

Requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation of a simian virus 40 early pre-RNA in vitro

1987 ◽  
Vol 7 (1) ◽  
pp. 495-503
Author(s):  
L C Ryner ◽  
J L Manley
Keyword(s):  
Simian Virus 40 ◽  
Nuclear Extract ◽  
Structural Features ◽  
Simian Virus ◽  
Micrococcal Nuclease ◽  
Small Nuclear Ribonucleoprotein ◽  
Cleavage And Polyadenylation ◽  
Nuclear Ribonucleoprotein ◽  
Cell Nuclear Extract

Using a pre-RNA containing the simian virus 40 early introns and poly(A) addition site, we investigated several possible requirements for accurate and efficient mRNA 3' end cleavage and polyadenylation in a HeLa cell nuclear extract. Splicing and 3' end formation occurred under the same conditions but did not appear to be coupled in any way in vitro. Like splicing, 3' end cleavage and polyadenylation each required Mg2+, although spermidine could substitute in the cleavage reaction. Additionally, cleavage of this pre-RNA, but not others, was totally blocked by EDTA, indicating that structural features of pre-RNA may affect the ionic requirements of 3' end formation. The ATP analog 3' dATP inhibited both cleavage and polyadenylation even in the presence of ATP, possibly reflecting the coupled nature of these activities. A 5' cap structure appears not to be required for mRNA 3' end processing in vitro because neither the presence or absence of a 5' cap on the pre-RNA nor the addition of cap analogs to reaction mixtures had any effect on the efficiency of 3' end processing. Micrococcal nuclease pretreatment of the nuclear extract inhibited cleavage and polyadenylation. However, restoration of activity was achieved by addition of purified Escherichia coli RNA, suggesting that the inhibition caused by such a nuclease treatment was due to a general requirement for mass of RNA rather than to destruction of a particular nucleic acid-containing component such as a small nuclear ribonucleoprotein.

Definition of the upstream efficiency element of the simian virus 40 late polyadenylation signal by using in vitro analyses

1992 ◽  
Vol 12 (12) ◽  
pp. 5386-5393 ◽  
Author(s):  
N Schek ◽  
C Cooke ◽  
J C Alwine
Keyword(s):  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Upstream Region ◽  
Core Element ◽  
The Core ◽  
Core Elements ◽  
Upstream Element

The polyadenylation signal for the late mRNAs of simian virus 40 is known to have sequence elements located both upstream and downstream of the AAUAAA which affect efficiency of utilization of the signal. The upstream efficiency element has been previously characterized by using deletion mutations and transfection analyses. Those studies suggested that the upstream element lies between 13 and 48 nucleotides upstream of the AAUAAA. We have utilized in vitro cleavage and polyadenylation reactions to further define the upstream element. 32P-labeled substrate RNAs were prepared by in vitro transcription from wild-type templates as well as from mutant templates having deletions and linker substitutions in the upstream region. Analysis of these substrates defined the upstream region as sequences between 13 and 51 nucleotides upstream of the AAUAAA, in good agreement with the in vivo results. Within this region, three core elements with the consensus sequence AUUUGURA were identified and were specifically mutated by linker substitution. These core elements were found to contain the active components of the upstream efficiency element. Using substrates with both single and double linker substitution mutations of core elements, we observed that the core elements function in a distance-dependent manner. In mutants containing only one core element, the effect on efficiency increases as the distance between the element and the AAUAAA decreases. In addition, when core elements are present in multiple copies, the effect is additive. The core element consensus sequence, which bears homology to the Sm protein complex-binding site in human U1 RNA, is also found within the upstream elements of the ground squirrel hepatitis B and cauliflower mosaic virus polyadenylation signals (R. Russnak, Nucleic Acids Res. 19:6449-6456, 1991; H. Sanfacon, P. Brodmann, and T. Hohn, Genes Dev. 5:141-149, 1991), suggesting functional conservation of this element between mammals and plants.

scholarly journals Cell cycle-regulated phosphorylation of the pre-mRNA-binding (heterogeneous nuclear ribonucleoprotein) C proteins.

1993 ◽  
Vol 13 (9) ◽  
pp. 5762-5770 ◽  
Author(s):  
S Piñol-Roma ◽  
G Dreyfuss
Keyword(s):  
Cell Cycle ◽  
Gel Electrophoresis ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Hnrnp C ◽  
C Proteins ◽  
Nuclear Ribonucleoprotein ◽  
Mrna Binding

Heterogeneous nuclear ribonucleoprotein (hnRNP) complexes, the structures that contain heterogeneous nuclear RNA and its associated proteins, constitute one of the most abundant components of the eukaryotic nucleus. hnRNPs appear to play important roles in the processing, and possibly also in the transport, of mRNA. hnRNP C proteins (C1, M(r) of 41,000; C2, M(r) of 43,000 [by sodium dodecyl sulfate-polyacrylamide gel electrophoresis]) are among the most abundant pre-mRNA-binding proteins, and they bind tenaciously to sequences relevant to pre-mRNA processing, including the polypyrimidine stretch of introns (when it is uridine rich). C proteins are found in the nucleus during the interphase, but during mitosis they disperse throughout the cell. They have been shown previously to be phosphorylated in vivo, and they can be phosphorylated in vitro by a casein kinase type II. We have identified and partially purified at least two additional C protein kinases. One of these, termed Cs kinase, caused a distinct mobility shift of C proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These phosphorylated C proteins, the Cs proteins, were the prevalent forms of C proteins during mitosis, and Cs kinase activity was also increased in extracts prepared from mitotic cells. Thus, hnRNP C proteins undergo cell cycle-dependent phosphorylation by a cell cycle-regulated protein kinase. Cs kinase activity appears to be distinct from the well-characterized mitosis-specific histone H1 kinase activity. Several additional hnRNP proteins are also phosphorylated during mitosis and are thus also potential substrates for Cs kinase. These novel phosphorylations may be important in regulating the assembly and disassembly of hnRNP complexes and in the function or cellular localization of RNA-binding proteins.

scholarly journals An RNA-binding protein specifically interacts with a functionally important domain of the downstream element of the simian virus 40 late polyadenylation signal.

1991 ◽  
Vol 11 (10) ◽  
pp. 5312-5320 ◽  
Author(s):  
Z W Qian ◽  
J Wilusz
Keyword(s):  
Binding Site ◽  
Rna Binding ◽  
Specific Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Band Shift

We have identified an RNA-binding protein which interacts with the downstream element of the simian virus 40 late polyadenylation signal in a sequence-specific manner. A partially purified 50-kDa protein, which we have named DSEF-1, retains RNA-binding specificity as assayed by band shift and UV cross-linking analyses. RNA footprinting assays, using end-labeled RNA ladder fragments in conjunction with native gel electrophoresis, have identified the DSEF-1 binding site as 5'-GGGGGAGGUGUGGG-3'. This 14-base sequence serves as an efficient DSEF-1 binding site when placed within a GEM4 polylinker-derived RNA. Finally, the DSEF-1 binding site restored efficient in vitro 3' end processing to derivatives of the simian virus 40 late polyadenylation signal in which it substituted for the entire downstream region. DSEF-1, therefore, may be a sequence-specific binding factor which regulates the efficiency of polyadenylation site usage.

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