Degradation of hammerhead ribozymes by human ribonucleases

L. Qiu, A. Moreira, G. Kaplan, R. Levitz, J. Y. Wang, C. Xu, K. Drlica

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Hammerhead ribozymes were used as substrates to examine endoribonucleolytic activities in cell extracts and cultured human cells. Primer-extension analyses showed that ribozymes directed against tumor necrosis factor-α mRNA and human immunodeficiency virus type 1 tat mRNA were cleaved at UA and CA dinucleotides by extracts. Preferred cleavage sites were similar to those observed following digestion with RNase A, and cleavage was blocked by RNasin, an inhibitor of pyrimidine-specific ribonucleases. Removal of UA and CA dinucleotides rendered ribozymes more stable when incubated in cell extracts that were not significantly contaminated by extracellular nucleases. Placement of UA dinucleotides adjacent to a ribozyme in mRNA led to excision of the ribozyme from long transcripts during incubation in extracts. UA dinucleotides also made mRNA more labile than a control RNA when expressed from an endogenous plasmid gene in the human myeloid cell line U937. Similarly, UA and CA dinucleotides caused ribozymes to have a shorter halflife when delivered to U937 cells by lipofectin-mediated transformation. Taken together, these data indicate that one or more members of the pyrimidine-specific ribonuclease family is involved in the intracellular degradation of RNA, and they explain the paucity of UA dinucleotides in eukaryotic mRNA. Judicious manipulation of preferred target sequences of pyrimidine-specific ribonucleases may be useful in designing effective hammerhead ribozymes.

Original languageEnglish (US)
Pages (from-to)352-362
Number of pages11
JournalMolecular and General Genetics
Volume258
Issue number4
DOIs
StatePublished - 1998

Fingerprint

Catalytic RNA
Ribonucleases
Messenger RNA
Cell Extracts
Pancreatic Ribonuclease
U937 Cells
RNA Stability
Myeloid Cells
Half-Life
HIV-1
Digestion
Cultured Cells
Plasmids
Tumor Necrosis Factor-alpha
hammerhead ribozyme
RNA
Cell Line
Genes
pyrimidine

Keywords

  • HIV-1 tat
  • Pyrimidine-specific ribonucleases
  • Tumor necrosis factor-α

ASJC Scopus subject areas

  • Genetics

Cite this

Qiu, L., Moreira, A., Kaplan, G., Levitz, R., Wang, J. Y., Xu, C., & Drlica, K. (1998). Degradation of hammerhead ribozymes by human ribonucleases. Molecular and General Genetics, 258(4), 352-362. https://doi.org/10.1007/s004380050741

Degradation of hammerhead ribozymes by human ribonucleases. / Qiu, L.; Moreira, A.; Kaplan, G.; Levitz, R.; Wang, J. Y.; Xu, C.; Drlica, K.

In: Molecular and General Genetics, Vol. 258, No. 4, 1998, p. 352-362.

Research output: Contribution to journalArticle

Qiu, L, Moreira, A, Kaplan, G, Levitz, R, Wang, JY, Xu, C & Drlica, K 1998, 'Degradation of hammerhead ribozymes by human ribonucleases', Molecular and General Genetics, vol. 258, no. 4, pp. 352-362. https://doi.org/10.1007/s004380050741
Qiu L, Moreira A, Kaplan G, Levitz R, Wang JY, Xu C et al. Degradation of hammerhead ribozymes by human ribonucleases. Molecular and General Genetics. 1998;258(4):352-362. https://doi.org/10.1007/s004380050741
Qiu, L. ; Moreira, A. ; Kaplan, G. ; Levitz, R. ; Wang, J. Y. ; Xu, C. ; Drlica, K. / Degradation of hammerhead ribozymes by human ribonucleases. In: Molecular and General Genetics. 1998 ; Vol. 258, No. 4. pp. 352-362.
@article{7ec456c93f0947168c588e09720dc3e1,
title = "Degradation of hammerhead ribozymes by human ribonucleases",
abstract = "Hammerhead ribozymes were used as substrates to examine endoribonucleolytic activities in cell extracts and cultured human cells. Primer-extension analyses showed that ribozymes directed against tumor necrosis factor-α mRNA and human immunodeficiency virus type 1 tat mRNA were cleaved at UA and CA dinucleotides by extracts. Preferred cleavage sites were similar to those observed following digestion with RNase A, and cleavage was blocked by RNasin, an inhibitor of pyrimidine-specific ribonucleases. Removal of UA and CA dinucleotides rendered ribozymes more stable when incubated in cell extracts that were not significantly contaminated by extracellular nucleases. Placement of UA dinucleotides adjacent to a ribozyme in mRNA led to excision of the ribozyme from long transcripts during incubation in extracts. UA dinucleotides also made mRNA more labile than a control RNA when expressed from an endogenous plasmid gene in the human myeloid cell line U937. Similarly, UA and CA dinucleotides caused ribozymes to have a shorter halflife when delivered to U937 cells by lipofectin-mediated transformation. Taken together, these data indicate that one or more members of the pyrimidine-specific ribonuclease family is involved in the intracellular degradation of RNA, and they explain the paucity of UA dinucleotides in eukaryotic mRNA. Judicious manipulation of preferred target sequences of pyrimidine-specific ribonucleases may be useful in designing effective hammerhead ribozymes.",
keywords = "HIV-1 tat, Pyrimidine-specific ribonucleases, Tumor necrosis factor-α",
author = "L. Qiu and A. Moreira and G. Kaplan and R. Levitz and Wang, {J. Y.} and C. Xu and K. Drlica",
year = "1998",
doi = "10.1007/s004380050741",
language = "English (US)",
volume = "258",
pages = "352--362",
journal = "Molecular Genetics and Genomics",
issn = "1617-4615",
publisher = "Springer Verlag",
number = "4",

}

TY - JOUR

T1 - Degradation of hammerhead ribozymes by human ribonucleases

AU - Qiu, L.

AU - Moreira, A.

AU - Kaplan, G.

AU - Levitz, R.

AU - Wang, J. Y.

AU - Xu, C.

AU - Drlica, K.

PY - 1998

Y1 - 1998

N2 - Hammerhead ribozymes were used as substrates to examine endoribonucleolytic activities in cell extracts and cultured human cells. Primer-extension analyses showed that ribozymes directed against tumor necrosis factor-α mRNA and human immunodeficiency virus type 1 tat mRNA were cleaved at UA and CA dinucleotides by extracts. Preferred cleavage sites were similar to those observed following digestion with RNase A, and cleavage was blocked by RNasin, an inhibitor of pyrimidine-specific ribonucleases. Removal of UA and CA dinucleotides rendered ribozymes more stable when incubated in cell extracts that were not significantly contaminated by extracellular nucleases. Placement of UA dinucleotides adjacent to a ribozyme in mRNA led to excision of the ribozyme from long transcripts during incubation in extracts. UA dinucleotides also made mRNA more labile than a control RNA when expressed from an endogenous plasmid gene in the human myeloid cell line U937. Similarly, UA and CA dinucleotides caused ribozymes to have a shorter halflife when delivered to U937 cells by lipofectin-mediated transformation. Taken together, these data indicate that one or more members of the pyrimidine-specific ribonuclease family is involved in the intracellular degradation of RNA, and they explain the paucity of UA dinucleotides in eukaryotic mRNA. Judicious manipulation of preferred target sequences of pyrimidine-specific ribonucleases may be useful in designing effective hammerhead ribozymes.

AB - Hammerhead ribozymes were used as substrates to examine endoribonucleolytic activities in cell extracts and cultured human cells. Primer-extension analyses showed that ribozymes directed against tumor necrosis factor-α mRNA and human immunodeficiency virus type 1 tat mRNA were cleaved at UA and CA dinucleotides by extracts. Preferred cleavage sites were similar to those observed following digestion with RNase A, and cleavage was blocked by RNasin, an inhibitor of pyrimidine-specific ribonucleases. Removal of UA and CA dinucleotides rendered ribozymes more stable when incubated in cell extracts that were not significantly contaminated by extracellular nucleases. Placement of UA dinucleotides adjacent to a ribozyme in mRNA led to excision of the ribozyme from long transcripts during incubation in extracts. UA dinucleotides also made mRNA more labile than a control RNA when expressed from an endogenous plasmid gene in the human myeloid cell line U937. Similarly, UA and CA dinucleotides caused ribozymes to have a shorter halflife when delivered to U937 cells by lipofectin-mediated transformation. Taken together, these data indicate that one or more members of the pyrimidine-specific ribonuclease family is involved in the intracellular degradation of RNA, and they explain the paucity of UA dinucleotides in eukaryotic mRNA. Judicious manipulation of preferred target sequences of pyrimidine-specific ribonucleases may be useful in designing effective hammerhead ribozymes.

KW - HIV-1 tat

KW - Pyrimidine-specific ribonucleases

KW - Tumor necrosis factor-α

UR - http://www.scopus.com/inward/record.url?scp=0031841806&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031841806&partnerID=8YFLogxK

U2 - 10.1007/s004380050741

DO - 10.1007/s004380050741

M3 - Article

VL - 258

SP - 352

EP - 362

JO - Molecular Genetics and Genomics

JF - Molecular Genetics and Genomics

SN - 1617-4615

IS - 4

ER -