Metal binding and substrate positioning by evolutionarily invariant U6 sequences in catalytically active protein-free snRNAs.

We have previously shown that a base-paired complex formed by two of the spliceosomal RNA components, U6 and U2 small nuclear RNAs (snRNAs), can catalyze a two-step splicing reaction that depended on an evolutionarily invariant region in U6, the ACAGAGA box. Here we further analyze this RNA-catalyzed reaction and show that while the 5' and 3' splice site substrates are juxtaposed and positioned near the ACAGAGA sequence in U6, the role of the snRNAs in the reaction is beyond mere juxtaposition of the substrates and likely involves the formation of a sophisticated active site. Interestingly, the snRNA-catalyzed reaction is metal dependent, as is the case with other known splicing RNA enzymes, and terbium(III) cleavage reactions indicate metal binding by the U6/U2 complex within the evolutionarily conserved regions of U6. The above results, combined with the structural similarities between U6 and catalytically critical domains in group II self-splicing introns, suggest that the base-paired complex of U6 and U2 snRNAs is a vestigial ribozyme and a likely descendant of a group II-like self-splicing intron.

Protein-free small nuclear RNAs catalyze a two-step splicing reaction.

Pre-mRNA splicing is a crucial step in eukaryotic gene expression and is carried out by a highly complex ribonucleoprotein assembly, the spliceosome. Many fundamental aspects of spliceosomal function, including the identity of catalytic domains, remain unknown. We show that a base-paired complex of U6 and U2 small nuclear RNAs, in the absence of the approximately 200 other spliceosomal components, performs a two-step reaction with two short RNA oligonucleotides as substrates that results in the formation of a linear RNA product containing portions of both oligonucleotides. This reaction, which is chemically identical to splicing, is dependent on and occurs in proximity of sequences known to be critical for splicing in vivo. These results prove that the complex formed by U6 and U2 RNAs is a ribozyme and can potentially carry out RNA-based catalysis in the spliceosome.

Treatment of Epstein-Barr Virus-Related Post-Renal Transplantation Development of Diffuse Large B Cell Lymphoma of the Distal Ureter: A Case Report.

Posttransplant lymphoproliferative disorder with involvement of the donor urogenital tissue is a rare and serious complication of solid organ transplant. We report an adult kidney transplant recipient who developed the diffuse large B cell lymphoma of the distal ureter in the setting of new allograft nephropathy. Early intervention, reduction of immunosuppression, surgical reconstruction and chemotherapy salvaged the allograft kidney and averted a fatal outcome. The renal function recovered to the baseline with creatinine ranging between 1.3 and 1.5. The patient did not require dialysis at any point after ureteral stent placement and reconstructive surgery. In addition, the case highlights the importance of multidisciplinary management involving transplant nephrology, oncology, transplant surgery, and urology in such a complicated disease process.

Splicing of an intervening sequence by protein-free human snRNAs.

Significant structural and mechanistic similarities between the spliceosomal snRNAs and catalytically critical domains of self-splicing group II introns have led to the hypothesis that the spliceosomes and group II introns may be evolutionarily related. We have previously shown that in vitro-transcribed, protein-free U6 and U2 snRNAs can catalyze a two-step splicing reaction in trans on two short RNA oligonucleotides that is identical to the splicing reactions performed by many self-splicing group II introns. Here we show that the same two snRNAs can perform splicing in cis by removal of an intervening sequence from a model substrate. These results prove that the protein-free snRNAs are competent to perform splicing on pre-mRNAs and further strengthen the possibility of an evolutionary relationship to group II introns.

The spliceosomal proteome: at the heart of the largest cellular ribonucleoprotein machine.

Almost all primary transcripts in higher eukaryotes undergo several splicing events and alternative splicing is a major factor in generating proteomic diversity. Thus, the spliceosome, the ribonucleoprotein assembly that performs splicing, is a highly critical cellular machine and as expected, a very complex one. Indeed, the spliceosome is one of the largest, if not the largest, molecular machine in the cell with over 150 different components in human. A large fraction of the spliceosomal proteome is organized into small nuclear ribonucleoprotein particles by associating with one of the small nuclear RNAs, and the function of many spliceosomal proteins revolve around their association or interaction with the spliceosomal RNAs or the substrate pre-messenger RNAs. In addition to the complex web of protein-RNA interactions, an equally complex network of protein-protein interactions exists in the spliceosome, which includes a number of large, conserved proteins with critical functions in the spliceosomal catalytic core. These include the largest conserved nuclear protein, Prp8, which plays a critical role in spliceosomal function in a hitherto unknown manner. Taken together, the large spliceosomal proteome and its dynamic nature has made it a highly challenging system to study, and at the same time, provides an exciting example of the evolution of a proteome around a backbone of primordial RNAs likely dating from the RNA World.

Low-dose heparin as treatment for early disseminated intravascular coagulation during sepsis: A prospective clinical study.

The present study aimed to investigate whether low-dose heparin improves the condition of patients suffering from early disseminated intravascular coagulation (pre-DIC) during sepsis. In total, 37 patients were randomly divided into low-dose heparin intervention and control groups. The heparin group received a low-dose of heparin for 5-7 days, while the other group received only saline. The two groups were treated for sepsis. Blood samples were collected at various times and acute physiology and chronic health evaluation (APACHE)-II scores were recorded at day 1 and 7. In addition, the number of days applying mechanical ventilation and in the intensive care unit (ICU) were recorded, as well as the 28-day mortality rate. APACHE-II scores in the two groups decreased following treatment, however, scores in the heparin group decreased more significantly. Prothrombin fragment and thrombin-antithrombin complex levels in the heparin group were significantly decreased. In addition, the number of days applying a ventilator was fewer and the total stay in ICU was significantly shorter compared with the control group. Significantly fewer complications were observed in the heparin group, however, there was no significant difference in the 28-day mortality rate. In conclusion, low-dose heparin improves the hypercoagulable state of sepsis, which subsequently reduces the incidence of DIC or multiple organ dysfunction syndrome, decreasing the number of days of mechanical ventilation and hospitalization.