scholarly journals The trypanocidal benzoxaborole AN7973 inhibits trypanosome mRNA processing

2018 ◽  
Author(s):  
Daniela Begolo ◽  
Isabel M Vincent ◽  
Federica Giordani ◽  
Michael J Witty ◽  
Timothy G Rowan ◽  
...  
Keyword(s):  
Mrna Processing ◽  
Granule Formation ◽  
Over Expression ◽  
Spliced Leader ◽  
Bovine Trypanosomosis ◽  
Cleavage And Polyadenylation ◽  
Adenosyl Methionine ◽  
Precursor Rna ◽  
Polyadenylation Factor ◽  
Selection Of

AbstractKinetoplastid parasites - trypanosomes and leishmanias - infect millions of humans and cause economically devastating diseases of livestock, and the few existing drugs have serious deficiencies. Benzoxaborole-based compounds are very promising potential novel anti-trypanosomal therapies, with candidates already in human and animal clinical trials. Their targets in trypanosomes were hitherto unknown. We investigated the mechanism of action of several benzoxaboroles, including AN7973, an early candidate for veterinary trypanosomosis.In all kinetoplastids, transcription is polycistronic. Individual mRNA 5’-ends are created by trans splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of Trypanosoma brucei with AN7973 inhibited trans splicing within 1h, as judged by loss of the Y-structure splicing intermediate and reduced levels of mRNA, and accumulation of peri-nuclear granules which are typical for splicing inhibition. Methylation of the spliced leader precursor RNA was not affected, but more prolonged AN7973 treatment caused an increase in S-adenosyl methionine and methylated lysine. Together, these results indicate that mRNA processing is the primary target of AN7973. Polyadenylation is required for kinetoplastid trans splicing. The EC50 for AN7973 in T. brucei was increased three-fold by over-expression of the T. brucei cleavage and polyadenylation factor CPSF3, identifying CPSF3 as a potential molecular target. Our results thus chemically validate mRNA processing as a viable drug target in trypanosomes.Several other benzoxaboroles showed metabolomic and splicing effects that were similar to those of AN7973, identifying splicing inhibition as a common mode of action, and suggesting that it might be linked to subsequent changes in methylated metabolites. Granule formation, splicing inhibition, and resistance after CPSF3 expression did not, however, always correlate, and prolonged selection of trypanosomes in AN7973 resulted in only 1.5-fold resistance. This suggests that the modes of action of oxaboroles that target trypanosome mRNA processing may extend beyond CPSF3 inhibition.Author summaryTrypanosomes and leishmanias infect millions of humans and cause economically devastating diseases of livestock; the few existing drugs have serious deficiencies. Trypanosomosis of cattle, caused mainly by Trypanosoma congolense and Trypanosoma vivax, is a serious problem in Africa, because bovids are used not only for meat and milk, but also for traction. Only two drugs are in routine use for chemotherapy and chemoprophylaxis of bovine trypanosomosis. A single injection of the benzoxaborole compound AN7973 was sufficient to cure T. congolense infection in cattle and goats, but AN7973 was less effective against T. vivax. This precluded development of AN7973 as a commercially viable treatment against cattle trypanosomosis, but it could still have potential for diseases caused by other salivarian trypanosomes.We used a large range of methods to find out how AN7973 kills trypanosomes, and compared it with several other benzoxaboroles. AN7973 and some of the other compounds had effects on parasite metabolism that resembled those previously seen for a benzoxaborole that is being tested for human sleeping sickness. The most rapid effect of AN7973, however, was on processing of trypanosome mRNA. As a consequence, amounts of mRNA decreased and synthesis of proteins stopped. We conclude that AN7973 and some other benzoxaboroles kill trypanosomes by stopping gene expression.

Separation of factors required for cleavage and polyadenylation of yeast pre-mRNA

1992 ◽  
Vol 12 (8) ◽  
pp. 3470-3481
Author(s):  
J Chen ◽  
C Moore
Keyword(s):  
Cell Extract ◽  
Yeast Cells ◽  
Factor I ◽  
Initial Separation ◽  
Cleavage And Polyadenylation ◽  
Precursor Rna ◽  
A Site ◽  
Processing Factors ◽  
Polyadenylation Factor ◽  
Mammalian System

Cleavage and polyadenylation of yeast precursor RNA require at least four functionally distinct factors (cleavage factor I [CF I], CF II, polyadenylation factor I [PF I], and poly(A) polymerase [PAP]) obtained from yeast whole cell extract. Cleavage of precursor occurs upon combination of the CF I and CF II fractions. The cleavage reaction proceeds in the absence of PAP or PF I. The cleavage factors exhibit low but detectable activity without exogenous ATP but are stimulated when this cofactor is included in the reaction. Cleavage by CF I and CF II is dependent on the presence of a (UA)6 sequence upstream of the GAL7 poly(A) site. The factors will also efficiently cleave precursor with the CYC1 poly(A) site. This RNA does not contain a UA repeat, and processing at this site is thought to be directed by a UAG...UAUGUA-type motif. Specific polyadenylation of a precleaved GAL7 RNA requires CF I, PF I, and a crude fraction containing PAP activity. The PAP fraction can be replaced by recombinant PAP, indicating that this enzyme is the only factor in this fraction needed for the reconstituted reaction. The poly(A) addition step is also dependent on the UA repeat. Since CF I is the only factor necessary for both cleavage and poly(A) addition, it is likely that this fraction contains a component which recognizes processing signals located upstream of the poly(A) site. The initial separation of processing factors in yeast cells suggests both interesting differences from and similarities to the mammalian system.

scholarly journals Intercistronic Region Required for Polycistronic Pre-mRNA Processing in Caenorhabditis elegans

2001 ◽  
Vol 21 (4) ◽  
pp. 1111-1120 ◽  
Author(s):  
Tao Huang ◽  
Scott Kuersten ◽  
Atul M. Deshpande ◽  
John Spieth ◽  
Margaret MacMorris ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Heat Shock ◽  
Mutational Analysis ◽  
Mrna Processing ◽  
Mrna Accumulation ◽  
Spliced Leader ◽  
Primary Signal ◽  
Cleavage And Polyadenylation

ABSTRACT In Caenorhabditis elegans, polycistronic pre-mRNAs are processed by cleavage and polyadenylation at the 3′ ends of the upstream genes and trans splicing, generally to the specialized spliced leader SL2, at the 5′ ends of the downstream genes. Previous studies have indicated a relationship between these two events in the processing of a heat shock-induced gpd-2–gpd-3polycistronic pre-mRNA. Here, we report mutational analysis of the intercistronic region of this operon by linker scan analysis. Surprisingly, no sequences downstream of the 3′ end were important for 3′-end formation. In contrast, a U-rich (Ur) element located 29 bp downstream of the site of 3′-end formation was shown to be important for downstream mRNA biosynthesis. This ∼20-bp element is sufficient for SL2 trans splicing and mRNA accumulation when transplanted to a heterologous context. Furthermore, when the downstream gene was replaced by a gene from another organism, no loss of trans-splicing specificity was observed, suggesting that the Ur element may be the primary signal required for downstream mRNA processing.

scholarly journals Separation of factors required for cleavage and polyadenylation of yeast pre-mRNA.

1992 ◽  
Vol 12 (8) ◽  
pp. 3470-3481 ◽  
Author(s):  
J Chen ◽  
C Moore
Keyword(s):  
Cell Extract ◽  
Yeast Cells ◽  
Factor I ◽  
Initial Separation ◽  
Cleavage And Polyadenylation ◽  
Precursor Rna ◽  
A Site ◽  
Processing Factors ◽  
Polyadenylation Factor ◽  
Mammalian System

Cleavage and polyadenylation of yeast precursor RNA require at least four functionally distinct factors (cleavage factor I [CF I], CF II, polyadenylation factor I [PF I], and poly(A) polymerase [PAP]) obtained from yeast whole cell extract. Cleavage of precursor occurs upon combination of the CF I and CF II fractions. The cleavage reaction proceeds in the absence of PAP or PF I. The cleavage factors exhibit low but detectable activity without exogenous ATP but are stimulated when this cofactor is included in the reaction. Cleavage by CF I and CF II is dependent on the presence of a (UA)6 sequence upstream of the GAL7 poly(A) site. The factors will also efficiently cleave precursor with the CYC1 poly(A) site. This RNA does not contain a UA repeat, and processing at this site is thought to be directed by a UAG...UAUGUA-type motif. Specific polyadenylation of a precleaved GAL7 RNA requires CF I, PF I, and a crude fraction containing PAP activity. The PAP fraction can be replaced by recombinant PAP, indicating that this enzyme is the only factor in this fraction needed for the reconstituted reaction. The poly(A) addition step is also dependent on the UA repeat. Since CF I is the only factor necessary for both cleavage and poly(A) addition, it is likely that this fraction contains a component which recognizes processing signals located upstream of the poly(A) site. The initial separation of processing factors in yeast cells suggests both interesting differences from and similarities to the mammalian system.

scholarly journals Cofactors are Remnants of Life’s Origin and Early Evolution

2021 ◽  
Vol 89 (3) ◽  
pp. 127-133 ◽  
Author(s):  
Aaron D. Goldman ◽  
Betul Kacar
Keyword(s):  
Genetic Information ◽  
Evolutionary Biology ◽  
Active Sites ◽  
Rna World ◽  
Genetic System ◽  
Early Evolution ◽  
Modern Biology ◽  
Iron Sulfur Cluster ◽  
Adenosyl Methionine ◽  
Precursor Rna

AbstractThe RNA World is one of the most widely accepted hypotheses explaining the origin of the genetic system used by all organisms today. It proposes that the tripartite system of DNA, RNA, and proteins was preceded by one consisting solely of RNA, which both stored genetic information and performed the molecular functions encoded by that genetic information. Current research into a potential RNA World revolves around the catalytic properties of RNA-based enzymes, or ribozymes. Well before the discovery of ribozymes, Harold White proposed that evidence for a precursor RNA world could be found within modern proteins in the form of coenzymes, the majority of which contain nucleobases or nucleoside moieties, such as Coenzyme A and S-adenosyl methionine, or are themselves nucleotides, such as ATP and NADH (a dinucleotide). These coenzymes, White suggested, had been the catalytic active sites of ancient ribozymes, which transitioned to their current forms after the surrounding ribozyme scaffolds had been replaced by protein apoenzymes during the evolution of translation. Since its proposal four decades ago, this groundbreaking hypothesis has garnered support from several different research disciplines and motivated similar hypotheses about other classes of cofactors, most notably iron-sulfur cluster cofactors as remnants of the geochemical setting of the origin of life. Evidence from prebiotic geochemistry, ribozyme biochemistry, and evolutionary biology, increasingly supports these hypotheses. Certain coenzymes and cofactors may bridge modern biology with the past and can thus provide insights into the elusive and poorly-recorded period of the origin and early evolution of life.

Poly(A) polymerase purified from HeLa cell nuclear extract is required for both cleavage and polyadenylation of pre-mRNA in vitro

1989 ◽  
Vol 9 (1) ◽  
pp. 193-203
Author(s):  
G Christofori ◽  
W Keller
Keyword(s):  
Hela Cell ◽  
Nuclear Extract ◽  
Cleavage Activity ◽  
Hela Cell Nuclear Extract ◽  
Cleavage And Polyadenylation ◽  
A Site ◽  
Polyadenylation Factor ◽  
Polyadenylated Mrna ◽  
Cell Nuclear Extract

We have partially purified a poly(A) polymerase (PAP) from HeLa cell nuclear extract which is involved in the 3'-end formation of polyadenylated mRNA. PAP had a molecular weight of approximately 50 to 60 kilodaltons. In the presence of manganese ions, PAP was able to polyadenylate RNA nonspecifically. However, in the presence of magnesium ions PAP required the addition of a cleavage and polyadenylation factor to specifically polyadenylate pre-mRNAs that contain an intact AAUAAA sequence and end at the poly(A) addition site (precleaved RNA substrates). The purified fraction containing PAP was also required in combination with a cleavage and polyadenylation factor and a cleavage factor for the correct cleavage at the poly(A) site of pre-mRNAs. Since the two activities of the PAP fractions, PAP and cleavage activity, could not be separated by extensive purification, we concluded that the two activities are contained in a single component, a PAP that is also required for the specific cleavage preceding the polyadenylation of pre-mRNA.

scholarly journals Identification of Factors Regulating Poly(A) Tail Synthesis and Maturation

2004 ◽  
Vol 24 (10) ◽  
pp. 4196-4206 ◽  
Author(s):  
David A. Mangus ◽  
Mandy M. Smith ◽  
Jennifer M. McSweeney ◽  
Allan Jacobson
Keyword(s):  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Genes Encoding ◽  
Mitogen Activated Protein ◽  
Regulatory Complex ◽  
Cleavage And Polyadenylation ◽  
Mrna Polyadenylation ◽  
Two Hybrid ◽  
Polyadenylation Factor

ABSTRACT Posttranscriptional maturation of the 3′ end of eukaryotic pre-mRNAs occurs as a three-step pathway involving site-specific cleavage, polymerization of a poly(A) tail, and trimming of the newly synthesized tail to its mature length. While most of the factors essential for catalyzing these reactions have been identified, those that regulate them remain to be characterized. Previously, we demonstrated that the yeast protein Pbp1p associates with poly(A)-binding protein (Pab1p) and controls the extent of mRNA polyadenylation. To further elucidate the function of Pbp1p, we conducted a two-hybrid screen to identify factors with which it interacts. Five genes encoding putative Pbp1p-interacting proteins were identified, including (i) FIR1/PIP1 and UFD1/PIP3, genes encoding factors previously implicated in mRNA 3′-end processing; (ii) PBP1 itself, confirming directed two-hybrid results and suggesting that Pbp1p can multimerize; (iii) DIG1, encoding a mitogen-activated protein kinase-associated protein; and (iv) PBP4 (YDL053C), a previously uncharacterized gene. In vitro polyadenylation reactions utilizing extracts derived from fir1Δ and pbp1Δ cells and from cells lacking the Fir1p interactor, Ref2p, demonstrated that Pbp1p, Fir1p, and Ref2p are all required for the formation of a normal-length poly(A) tail on precleaved CYC1 pre-mRNA. Kinetic analyses of the respective polyadenylation reactions indicated that Pbp1p is a negative regulator of poly(A) nuclease (PAN) activity and that Fir1p and Ref2p are, respectively, a positive regulator and a negative regulator of poly(A) synthesis. We suggest a model in which these three factors and Ufd1p are part of a regulatory complex that exploits Pab1p to link cleavage and polyadenylation factors of CFIA and CFIB (cleavage factors IA and IB) to the polyadenylation factors of CPF (cleavage and polyadenylation factor).

scholarly journals The Polyadenylation Factor CPSF-73 Is Involved in Histone-Pre-mRNA Processing

Cell ◽  
2005 ◽  
Vol 123 (1) ◽  
pp. 37-48 ◽  
Author(s):  
Zbigniew Dominski ◽  
Xiao-cui Yang ◽  
William F. Marzluff
Keyword(s):  
Mrna Processing ◽  
Polyadenylation Factor

scholarly journals Direct Interactions between the Paf1 Complex and a Cleavage and Polyadenylation Factor Are Revealed by Dissociation of Paf1 from RNA Polymerase II

2008 ◽  
Vol 7 (7) ◽  
pp. 1158-1167 ◽  
Author(s):  
Kristen Nordick ◽  
Matthew G. Hoffman ◽  
Joan L. Betz ◽  
Judith A. Jaehning
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Direct Interaction ◽  
Pol Ii ◽  
Phosphorylated Form ◽  
Cleavage And Polyadenylation ◽  
Paf1 Complex ◽  
Processing Factors ◽  
Polyadenylation Factor ◽  
Transcription Cycle

ABSTRACT The Paf1 complex (Paf1, Ctr9, Cdc73, Rtf1, and Leo1) is normally associated with RNA polymerase II (Pol II) throughout the transcription cycle. However, the loss of either Rtf1 or Cdc73 results in the detachment of the Paf1 complex from Pol II and the chromatin form of actively transcribed genes. Using functionally tagged forms of the Paf1 complex factors, we have determined that, except for the more loosely associated Rtf1, the remaining components stay stably associated with one another in an RNase-resistant complex after dissociation from Pol II and chromatin. The loss of Paf1, Ctr9, or to a lesser extent Cdc73 or Rtf1 results in reduced levels of serine 2 phosphorylation of the Pol II C-terminal domain and in increased read through of the MAK21 polyadenylation site. We found that the cleavage and polyadenylation factor Cft1 requires the Pol II-associated form of the Paf1 complex for full levels of interaction with the serine 5-phosphorylated form of Pol II. When the Paf1 complex is dissociated from Pol II, a direct interaction between Cft1 and the Paf1 complex can be detected. These results are consistent with the Paf1 complex providing a point of contact for recruitment of 3′-end processing factors at an early point in the transcription cycle. The lack of this connection helps to explain the defects in 3′-end formation observed in the absence of Paf1.

Sign in / Sign up

Export Citation Format

Share Document