Gene expression in the mitochondria of the kinetoplastid parasite Trypanosoma brucei is regulated primarily post-transcriptionally at the stages of RNA processing, editing, and turnover. The mitochondrial RNA-binding complex 1 (MRB1) is a recently identified multiprotein complex containing components with distinct functions during different aspects of RNA metabolism, such as guide RNA (gRNA) and mRNA turnover, precursor transcript processing, and RNA editing. In this study we examined the function of the MRB1 protein, Tb927.5.3010, which we term MRB3010. We show that MRB3010 is essential for growth of both procyclic form and bloodstream form life-cycle stages of T. brucei. Down-regulation of MRB3010 by RNAi leads to a dramatic inhibition of RNA editing, yet its depletion does not impact total gRNA levels. Rather, it appears to affect the editing process at an early stage, as indicated by the accumulation of pre-edited and small partially edited RNAs. MRB3010 is present in large (>20S) complexes and exhibits both RNA-dependent and RNA-independent interactions with other MRB1 complex proteins. Comparison of proteins isolated with MRB3010 tagged at its endogenous locus to those reported from other MRB1 complex purifications strongly suggests the presence of an MRB1 ''core'' complex containing five to six proteins, including MRB3010. Together, these data further our understanding of the function and composition of the imprecisely defined MRB1 complex.

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RNA turnover and RNA editing are essential for regulation of mitochondrial gene expression in Trypanosoma brucei. RNA turnover is controlled in part by RNA 3 adenylation and uridylation status, with trans-acting factors also impacting RNA homeostasis. However, little is known about the mitochondrial degradation machinery or its regulation in T. brucei. We have identified a mitochondrial exoribonuclease, TbRND, whose expression is highly up-regulated in the insect proliferative stage of the parasite. TbRND shares sequence similarity with RNase D family enzymes but differs from all reported members of this family in possessing a CCHC zinc finger domain. In vitro, TbRND exhibits 3 to 5 exoribonuclease activity, with specificity toward uridine homopolymers, including the 3 oligo(U) tails of guide RNAs (gRNAs) that provide the sequence information for RNA editing. Several lines of evidence generated from RNAimediated knockdown and overexpression cell lines indicate that TbRND functions in gRNA metabolism in vivo. First, TbRND depletion results in gRNA tails extended by 2-3 nucleotides on average. Second, overexpression of wild type but not catalytically inactive TbRND results in a substantial decrease in the total gRNA population and a consequent inhibition of RNA editing. The observed effects on the gRNA population are specific as rRNAs, which are also 3-uridylated, are unaffected by TbRND depletion or overexpression. Finally, we show that gRNA binding proteins co-purify with TbRND. In summary, TbRND is a novel 3 to 5 exoribonuclease that appears to have evolved a function highly specific to the mitochondrion of trypanosomes.Recognition of the importance of post-transcriptional processes has changed the way we look at gene regulation (1). RNA degradation that occurs in both substrate processing and turnover is an important aspect of post-transcriptional regulation in all organisms (1-7), and the factors controlling these processes are beginning to be elucidated. Increasingly, we find that 3Ј non-encoded tails serve as cis-acting elements in RNA stability regulation. Perhaps the most commonly reported examples of this are oligo(A) tails acting in both RNA stabilization and destabilization (5, 8 -14). However, oligo(U) tails have also been found to destabilize microRNAs, siRNAs, and decay intermediates and possibly act as quality control mechanisms (15-20). Non-encoded 3Ј tails can recruit and influence the activity of exoribonucleases, which along with endoribonucleases, supply the catalytic activity necessary for RNA degradation. Exoribonucleases are organized into a number of different families or classes (21). Some of these enzymes, such as RNase II, PNPase, and Xrn1, exhibit processive activity and participate in overall RNA turnover pathways, although each is a member of a different family. Others, like RNase D, a member of the DEDD class of exoribonucleases, have distributive activity and function in very specific regulatory roles (21-23). In organelles, regulation of RNA stability and the classes of exoribonuc...

A successful outcome after CFT is dependent on instilling biopsychosocial pain beliefs and developing independence among participants. Small improvers may require ongoing support to maintain results. Further study is needed to elucidate the optimal approach for those who were unchanged.

Summary Expression of class I human leucocyte antigens (HLA) on the surface of malignant cells is critical for their recognition and destruction by cytotoxic T lymphocytes. Surface expression requires assembly and folding of HLA class I molecules in the endoplasmic reticulum with the assistance of proteins such as Transporter associated with Antigen Processing (TAP) and tapasin. Interferon‐γ induces both TAP and tapasin so dissection of which protein contributes more to HLA class I expression has not been possible previously. In this study, we take advantage of a human melanoma cell line in which TAP can be induced, but tapasin cannot. Interferon‐γ increases TAP protein levels dramatically but HLA class I expression at the cell surface does not increase substantially, indicating that a large increase in peptide supply is not sufficient to increase HLA class I expression. On the other hand, transfection of either allelic form of tapasin (R240 or T240) enhances HLA‐B*5001 and HLA‐B*5701 antigen expression considerably with only a modest increase in TAP. Together, these data indicate that in the presence of minimal TAP activity, tapasin can promote substantial HLA class I expression at the cell surface.