Laura N. Rusché

Purification of a functional enzymatic editing complex from Trypanosoma brucei mitochondria

Kinetoplastid mitochondrial RNA editing, the insertion and deletion of U residues, is catalyzed by sequential cleavage, U addition or removal, and ligation reactions and is directed by complementary guide RNAs. We have purified a ∼20S enzymatic complex from Trypanosoma bruceimitochondria that catalyzes a complete editing reaction in vitro. This complex possesses all four activities predicted to catalyze RNA editing: gRNA‐directed endonuclease, terminal uridylyl transferase, 3′ U‐specific exonuclease, and RNA ligase. However, it does not contain other putative editing complex components: gRNA‐independent endonuclease, RNA helicase, endogenous gRNAs or pre‐mRNAs, or a 25 kDa gRNA‐binding protein. The complex is composed of eight major polypeptides, three of which represent RNA ligase. These findings identify polypeptides representing catalytic editing factors, reveal the nature of this ∼20S editing complex, and suggest a new model of editosome assembly.

The Two RNA Ligases of the Trypanosoma brucei RNA Editing Complex: Cloning the Essential Band IV Gene and Identifying the Band V Gene

ABSTRACT Kinetoplastid RNA editing is a posttranscriptional insertion and deletion of U residues in mitochondrial transcripts that involves RNA ligase. A complex of seven different polypeptides purified fromTrypanosoma brucei mitochondria that catalyzes accurate RNA editing contains RNA ligases of ∼57 kDa (band IV) and ∼50 kDa (band V). From a partial amino acid sequence, cDNA and genomic clones of band IV were isolated, making it the first cloned component of the minimal RNA editing complex. It is indeed an RNA ligase, for when expressed inEscherichia coli, the protein autoadenylylates and catalyzes RNA joining. Overexpression studies revealed that T. brucei can regulate of total band IV protein at the level of translation or protein stability, even upon massively increased mRNA levels. The proteins mitochondrial targeting was confirmed by its location, size when expressed in T. brucei and E. coli, and N-terminal sequence. Importantly, genetic knockout studies demonstrated that the gene for band IV is essential in procyclic trypanosomes. The band IV and band V RNA ligases of the RNA editing complex therefore serve different functions. We also identified the gene for band V RNA ligase, a protein much more homologous to band IV than to other known ligases.

Trypanosome RNA Editing: Simple Guide RNA Features Enhance U Deletion 100-Fold

ABSTRACT Trypanosome RNA editing is a massive processing of mRNA by U deletion and U insertion, directed by trans-acting guide RNAs (gRNAs). A U deletion cycle and a U insertion cycle have been reproduced in vitro using synthetic ATPase (A6) pre-mRNA and gRNA. Here we examine which gRNA features are important for this U deletion. We find that, foremost, this editing depends critically on the single-stranded character of a few gRNA and a few mRNA residues abutting the anchor duplex, a feature not previously appreciated. That plus any base-pairing sequence to tether the upstream mRNA are all the gRNA needs to direct unexpectedly efficient in vitro U deletion, using either the purified editing complex or whole extract. In fact, our optimized gRNA constructs support faithful U deletion up to 100 times more efficiently than the natural gRNA, and they can edit the majority of mRNA molecules. This is a marked improvement of in vitro U deletion, in which previous artificial gRNAs were no more active than natural gRNA and the editing efficiencies were at most a few percent. Furthermore, this editing is not stimulated by most other previously noted gRNA features, including its potential ligation bridge, 3′ OH moiety, any U residues in the tether, the conserved structure of the central region, or proteins that normally bind these regions. Our data also have implications about evolutionary forces active in RNA editing.

Direct Sizing of RNA Fragments Using RNase-Generated Standards

Publisher Summary Electrophoretic migration of RNA is affected by its terminal OH or P character to a sizable and variable extent, considerably more than is DNA. If not accounted for when sizing RNA, errors of up to two nucleotides can arise, even when using sequencing ladders prepared from RNA with the same sequence. Many cellular cleavage events of interest generate 3′-OH termini, yet these termini are not formed when using the standard methods of preparing RNA sequencing ladders, which all generate 2′,3′-cyclic P and 5′-OH termini. Fortunately, conditions have been found in which nuclease P1 cleaves in a highly G-specific manner and different conditions in which it cleaves in an A-preferential manner, allowing convenient preparation of the desired sequencing ladders. The key to accurate RNA size determination is to select appropriate marker ladders that not only are from the same sequence RNA but also have comparable termini. Cleavage agents commonly used to generate such ladders are sequence-specific RNases, including RNase T1 (which cleaves after G residues) and RNase A (which cleaves after C and U residues), and alkali, which cleaves after any residue.