Genome-Wide Determination of Poly(A) Site Choice in Plants

Author(s):  
Pratap Kumar Pati ◽  
Liuyin Ma ◽  
Arthur G. Hunt
Keyword(s):  
2020 ◽  
Vol 402 (1) ◽  
pp. 89-98
Author(s):  
Nathalie Meiser ◽  
Nicole Mench ◽  
Martin Hengesbach

AbstractN6-methyladenosine (m6A) is the most abundant modification in mRNA. The core of the human N6-methyltransferase complex (MTC) is formed by a heterodimer consisting of METTL3 and METTL14, which specifically catalyzes m6A formation within an RRACH sequence context. Using recombinant proteins in a site-specific methylation assay that allows determination of quantitative methylation yields, our results show that this complex methylates its target RNAs not only sequence but also secondary structure dependent. Furthermore, we demonstrate the role of specific protein domains on both RNA binding and substrate turnover, focusing on postulated RNA binding elements. Our results show that one zinc finger motif within the complex is sufficient to bind RNA, however, both zinc fingers are required for methylation activity. We show that the N-terminal domain of METTL3 alters the secondary structure dependence of methylation yields. Our results demonstrate that a cooperative effect of all RNA-binding elements in the METTL3–METTL14 complex is required for efficient catalysis, and that binding of further proteins affecting the NTD of METTL3 may regulate substrate specificity.


PLoS ONE ◽  
2017 ◽  
Vol 12 (1) ◽  
pp. e0171088 ◽  
Author(s):  
George Msalya ◽  
Eui-Soo Kim ◽  
Emmanuel L. K. Laisser ◽  
Maulilio J. Kipanyula ◽  
Esron D. Karimuribo ◽  
...  

2021 ◽  
Author(s):  
Lijie Wang ◽  
Wei Xue ◽  
Hongxia Zhang ◽  
Runze Gao ◽  
Houyuan Qiu ◽  
...  

Abstract Fusion of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) for programmable C-to-T editing, which holds potentials in clinical applications but suffers from off-target (OT) mutations. Here, we applied a cleavable deoxycytidine deaminase inhibitor (dCDI) domain to construct a transformer BE (tBE) system that induces efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. This step-by-step protocol describes the plasmid construction of tBE system, determination of genome/transcriptome-wide OT mutations and tBE-mediated base editing in vivo.


2016 ◽  
Vol 214 (1) ◽  
pp. 257-270 ◽  
Author(s):  
Zifeng Guo ◽  
Dijun Chen ◽  
Ahmad M. Alqudah ◽  
Marion S. Röder ◽  
Martin W. Ganal ◽  
...  

Author(s):  
Kara Todd ◽  
Freyja Brandel-Tanis ◽  
Daniel Arias ◽  
Kari Edison Watkins

As transit agencies expand, they may outgrow their existing bus storage and service facilities. When selecting a site for an additional facility, an important consideration is the change in bus deadhead time, which affects the agency’s operating costs. Minimizing bus deadhead time is the subject of many studies, though agencies may lack the necessary software or programming skill to implement those methods. This study presents a flexible tool for determination of bus facility location. Using the R dodgr package, it evaluates each candidate site based on a given bus network and existing depots and calculates the network minimum deadhead time for each potential set of facilities. Importantly, the tool could be used by any transit agency, no matter its resources. It runs on open-source software and uses only General Transit Feed Specification (GTFS) and data inputs readily available to transit agencies in the U.S.A., filling the accessibility gap identified in the literature. The tool is demonstrated through a case study with the Metropolitan Atlanta Rapid Transit Authority (MARTA), which is considering a new bus depot as it builds its bus rapid transit network. The case study used current MARTA bus GTFS data, existing depot locations, and vacant properties from Fulton County, Georgia. The tool evaluated 17 candidate sites and found that the winning site would save 29.7 deadhead hours on a typical weekday, which translates to more than $12,000 daily based on operating cost assumptions. The output provides important guidance to transit agencies evaluating sites for a new bus depot.


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