Characterization of a trna interacting component of the Leishmania mitochondrial tRNA import complex

There is a remarkable diversity in the scope and mechanism of mitochondrial tRNA import (reviewed in reference 268).Human mitochondria do not import tRNA, but a number of neuromuscular degenerative and metabolic diseases are caused by mutations in mitochondrial tRNA genes (321).In yeast a single tRNA is imported,apparently through protein import channels and requiring at least two soluble factors, including the mitochondrial form of the cognate aminoacyl-tRNA synthetase(274).By contrast , in kinetoplastid protozoa (Leishmania and trypanosomes),import of a whole spectrum of tRNAs is necessitated by the complete lack of mitochondrial tRNA genes(277,322). In this system,membrane-bound tRNAbinding proteins recognize specific structural motifs(import signals) on tRNA, soluble factors are not required , and the translocation pathway appears to be distinct from that of protein import(293,302,323).Moreover, the sequence and bioenergetic requirements for outer and inner membrane transfer are nonidentical(304),indicating the presence of a distinct transport machinery(theRNA import complex [RIC]) at the inner membrane, a situation similar to the TOM and TIM complexes for protein import (324).A 15-kDa polypeptide has been shown to be required for import into Leishmania mitochondria(295); otherwise,the import machinery remains undefined. Using an in vitro evolution protocol, it was recently shown that Leishmania mitochondria recognize a number of short sequence motifs homologous to multiple domains in tRNAs, suggesting the presence of several import signals (305).Moreover, novel positive and negative allosteric interactions between these aptamers , as well as between intact tRNAs, at the inner membrane were described (305).The RNAs could be classified into two types: type I RNAs are efficiently transferred through the inner membrane but are inhibited by type II . In contrast, type II RNAs have poor inner membrane transfer efficiencies and are stimulated by type I . For example, tRNATyr (GUA) is a type I RNA containing the conserved motif UAGAGC in the D domain , while tRNAIle (UAU) is type II with the sequence UCGCGGGUU in the variable loop-T domain (V-T) region(305).The mechanism of these allosteric interactions is unknown, but there are several possibilities . A single conformationally flexible dimeric or multimeric receptor could bind to either a type I or a type II motif. Alternatively, distinct type I and type II receptors may interact directly or indirectly through a third subunit. A related issue is whether the effector and substrate binding subunits for either RNA are identical or different (306). 2 To begin to define the molecular components of the import machinery , the group has recently reported the isolation of a multi-protein complex (the RNA Import Complex, or RIC) that is sufficient to induce import of tRNAs into artificial phospholipid vesicles (268). This reconstituted system retains all the properties of import in intact mitoplasts, including ATP dependence, and sensitivity to respiratory uncouplers and inhibitors (306). Two tRNA-binding proteins were identified within this complex by photo-crosslinking and immunochemistry: a 45-kDa protein that binds tRNATyr directly , and another 21-kDa protein that binds tRNAIle only in the presence of tRNATyr, suggesting allosteric changes within RIC leading to modulation of tRNA affinities (305-307). The identities of these tRNA-binding proteins are presently unknown. Ongoing work in the laboratory suggests that the 45-kDa band of RIC resolved by SDS-PAGE contains more than one protein species .E xpression of these open reading frames (ORFs) in bacteria , and the generation of ORF –specific antibodies , is in progress. These reagents will be useful in the identification of the tRNA- binding component. In the light of the above, the objectives of the proposed research are: (i) To obtain and analyze the genes corresponding to the protein species present in the 45 kDa band of the Leishmania RNA Import Complex; (ii) To study the organization & expression of these genes in Leishmania; and (iii) To study the effect of conditional knockout of these genes on mitochondrial function in vivo

Necessary and sufficient factors for the import of transfer RNA into the kinetoplast mitochondrion

The mechanism of active transport of transfer RNA (tRNA) across membranes is largely unknown. Factors mediating the import of tRNA into the kinetoplast mitochondrion of the protozoon Leishmania tropica are organized into a multiprotein RNA import complex (RIC) at the inner membrane. Here, we present the complete characterization of the identities and functions of the subunits of this complex. The complex contains three mitochondrion- and eight nuclear-encoded subunits; six of the latter are necessary and sufficient for import. Antisense-mediated knockdown of essential subunits resulted in the depletion of mitochondrial tRNAs and inhibition of organellar translation. Functional complexes were reconstituted with recombinant subunits expressed in Escherichia coli. Several essential RIC subunits are identical to specific subunits of respiratory complexes. These findings provide new information on the evolution of tRNA import and the foundation for detailed structural and mechanistic studies

Society of Gastrointestinal Endoscopy of India Consensus Guidelines on Endoscopic Ultrasound-Guided Biliary Drainage: Part II (Technical Aspects)

Endoscopic management of bile duct obstruction is a key aspect in gastroenterology practice and has evolved since the first description of biliary cannulation by McCune et al in 1968. Over many decades, the techniques and accessories have been refined, and currently, the first-line management for extrahepatic biliary obstruction is endoscopic retrograde cholangiopancreatography (ERCP). However, even in expert hands, the success rate of ERCP reaches up to 95%. In almost 4 to 16% cases, failure to cannulate the bile duct may necessitate other alternatives such as surgical bypass or, more commonly, percutaneous transhepatic biliary drainage (PTBD). While surgery is associated with high morbidity and mortality, PTBD has a very high reintervention and complication rate (∼80%) and poor quality of life. Almost parallelly, endoscopic ultrasound (EUS) has come a long way from a mere diagnostic tool to a substantial therapeutic option in various pancreaticobiliary diseases. Biliary drainage using EUS-guidance (EUS-BD) has gained momentum since the first report published by Giovannini et al in 2001. The concept of accessing the bile duct through a different route than the papilla, circumventing the shortcomings of PTBD, and sometimes bypassing the actual obstruction have enthused a lot of interest in this novel strategy. The three key methods of EUS-BD entail transluminal, antegrade, and rendezvous approach. Over the past decade, with growing experience, EUS-BD has been found to be equivalent to ERCP or PTBD for malignant obstruction with better success rates. EUS-BD, however, is not devoid of adverse events and can carry fatal adverse events. However, neither the technique of EUS-BD nor the accessories and stents for EUS-BD have been standardized. Additionally, different countries and regions have different availability of the accessories, making generalizability a difficult task. Thus, technical aspects of this evolving therapy need to be outlined. For these reasons, Society of Gastrointestinal Endoscopy of India (SGEI) deemed it appropriate to develop technical consensus statements for performing safe and successful EUS-BD

Society of Gastrointestinal Endoscopy of India Consensus Guidelines on Endoscopic Ultrasound-Guided Biliary Drainage: Part I (Indications, Outcomes, Comparative Evaluations, Training)

Endoscopic management of bile duct obstruction is a key aspect in gastroenterology practice and has evolved since the first description of biliary cannulation by McCune et al in 1968. Over many decades, the techniques and accessories have been refined and currently, the first-line management for extrahepatic biliary obstruction is endoscopic retrograde cholangiopancreaticography (ERCP). However, even in expert hands the success rate of ERCP reaches up to 95%. In almost 4 to 16% cases, failure to cannulate the bile duct may necessitate other alternatives such as surgical bypass or more commonly percutaneous transhepatic biliary drainage (PTBD). While surgery is associated with high morbidity and mortality, PTBD has a very high reintervention and complication rate (∼80%) and poor quality of life. Almost parallelly, endoscopic ultrasound (EUS) has come a long way from a mere diagnostic tool to a substantial therapeutic option in various pancreatico-biliary diseases. Biliary drainage using EUS-guidance (EUS-BD) has gained momentum since the first report published by Giovannini et al in 2001. The concept of accessing the bile duct through a different route than the papilla, circumventing the shortcomings of PTBD and sometimes bypassing the actual obstruction have enthused a lot of interest in this novel strategy. The three key methods of EUS-BD entail transluminal, antegrade, and rendezvous approach. Over the past decade, with growing experience, EUS-BD has been found to be equivalent to ERCP or PTBD for malignant obstruction with better success rates. EUS-BD, albeit, is not devoid of adverse events and can carry fatal adverse events. However, neither the technique of EUS-BD, nor the accessories and stents for EUS-BD have been standardized. Additionally, different countries and regions have different availability of the accessories making generalizability a difficult task. Thus, technical aspects of this evolving therapy need to be outlined. For these reasons, the Society of Gastrointestinal Endoscopy India deemed it appropriate to develop technical consensus statements for performing safe and successful EUS-BD