Retention and splicing complex (RES) - the importance of cooperativity.

One of the great challenges to structural biologists lies in explaining the complexities of the spliceosome - a ribosome-sized molecular machine that is assembled in a step-wise manner and is responsible for pre-mRNA splicing. The spliceosome is both fascinating and difficult to work with, because of its dynamic nature. At each discrete step of splicing tens of proteins come and go orchestrating the functional transition through massive structural rearrangements. The retention and splicing complex (RES) is an important splicing factor interacting with pre-mRNA at the onset of the first transesterification reaction. RES is a specific splicing factor for a number of genes and is important for controlling pre-mRNA retention in the nucleus. RES is a 71kDa heterotrimer composed of the 3 proteins Pml1p, Bud13p and Snu17p. We solved the 3-dimensional structure of the core of the RES complex as well as the 2 dimers, Snu17p-Bud13p and Snu17p-Pml1p. Further biophysical analysis revealed an astounding cooperativity that governs the assembly of this trimeric complex as well as its interaction with pre-mRNA. The more than 100-fold cooperativity originates from the progressive rigidification of Snu17p upon coupled binding-and-folding of protein regions, which are disordered in the unbound state. Our work highlights the role of cooperativity in the spliceosome and poses new questions about the structure and assembly of the spliceosome

Structures of intermediates during RES complex assembly.

The action of the spliceosome depends on the stepwise cooperative assembly and disassembly of its components. Very strong cooperativity was observed for the RES (Retention and Splicing) hetero-trimeric complex where the affinity from binary to tertiary interactions changes more than 100-fold and affects RNA binding. The RES complex is involved in splicing regulation and retention of not properly spliced pre-mRNA with its three components-Snu17p, Pml1p and Bud13p-giving rise to the two possible intermediate dimeric complexes Pml1p-Snu17p and Bud13p-Snu17p. Here we determined the three-dimensional structure and dynamics of the Pml1p-Snu17p and Bud13p-Snu17p dimers using liquid state NMR. We demonstrate that localized as well as global changes occur along the RES trimer assembly pathway. The stepwise rigidification of the Snu17p structure following the binding of Pml1p and Bud13p provides a basis for the strong cooperative nature of RES complex assembly

( 210 Pb/ 226 Ra) variations during the 1994-2001 intracaldera volcanism at Rabaul Caldera

Determining the timing and source of gas transfer during intermittent intracaldera volcanism can aid in our understanding of degassing in these large systems. Using (210Pb/226Ra) ratios, (parentheses denote activity ratios) as a time-sensitive tracer, injections of 222Rn and the subsequent time scales of gas accumulation and loss can be determined. Variations in (210Pb/226Ra) have been measured for 15 volcanic products erupted at Rabaul Caldera over the period 1994 to 2001. In addition, one basaltic enclave from the 1937 eruption was also analyzed. Water and carbon dioxide contents determined from olivine hosted melt inclusions erupted in 1997 are < 1% and suggest extensive shallow-level degassing. Both 210Pb excesses and deficits are found in andesites and dacites, whereas the basaltic enclave displays an (210Pb/226Ra)0 ratio of 7. Between 1994 and 1997 three samples with (210Pb/226Ra) deficits were erupted which indicate open system gas loss since 1992 and 1994. No correlation exists between (210Pb/226Ra) and lava chemistry, eruptive style or date. 210Pb excesses are more common than deficits in Rabaul samples but cannot be explained by plagioclase feldspar accumulation, Pb sublimate accumulation or differentiation. Instead, a model of intra-magma 222Rn transfer can produce 210Pb excesses of the appropriate magnitude if gas transfer occurs over 1-5 years from an underlying body of magma that is 2-10 times larger than the volume of erupted material and that is consistent with geophysical estimates. Although intermittent gas transfer events can be inferred by the development of 210Pb excess, there is no evidence at Rabaul for a direct link between eruptive style, gas flux and (210Pb/226Ra)

Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex.

The precursor mRNA (pre-mRNA) retention and splicing (RES) complex is a spliceosomal complex that is present in yeast and humans and is important for RNA splicing and retention of unspliced pre-mRNA. Here, we present the solution NMR structure of the RES core complex from Saccharomyces cerevisiae. Complex formation leads to an intricate folding of three components-Snu17p, Bud13p and Pml1p-that stabilizes the RNA-recognition motif (RRM) fold of Snu17p and increases binding affinity in tertiary interactions between the components by more than 100-fold compared to that in binary interactions. RES interacts with pre-mRNA within the spliceosome, and through the assembly of the RES core complex RNA binding efficiency is increased. The three-dimensional structure of the RES core complex highlights the importance of cooperative folding and binding in the functional organization of the spliceosome

(²¹⁰Pb/²²⁶Ra) variations during the 1994-2001 intracaldera volcanism at Rabaul Caldera

Determining the timing and source of gas transfer during intermittent intracaldera volcanism can aid in our understanding of degassing in these large systems. Using (²¹⁰Pb/²²⁶Ra) ratios, (parentheses denote activity ratios) as a time-sensitive tracer, injections of ²²²Rn and the subsequent time scales of gas accumulation and loss can be determined. Variations in (²¹⁰Pb/²²⁶Ra) have been measured for 15 volcanic products erupted at Rabaul Caldera over the period 1994 to 2001. In addition, one basaltic enclave from the 1937 eruption was also analyzed. Water and carbon dioxide contents determined from olivine hosted melt inclusions erupted in 1997 are < 1% and suggest extensive shallow-level degassing. Both ²¹⁰Pb excesses and deficits are found in andesites and dacites, whereas the basaltic enclave displays an (²¹⁰Pb/²²⁶Ra)0 ratio of 7. Between 1994 and 1997 three samples with (²¹⁰Pb/²²⁶Ra) deficits were erupted which indicate open system gas loss since 1992 and 1994. No correlation exists between (²¹⁰Pb/²²⁶Ra) and lava chemistry, eruptive style or date. ²¹⁰Pb excesses are more common than deficits in Rabaul samples but cannot be explained by plagioclase feldspar accumulation, Pb sublimate accumulation or differentiation. Instead, a model of intra-magma ²²²Rn transfer can produce ²¹⁰Pb excesses of the appropriate magnitude if gas transfer occurs over 1–5 years from an underlying body of magma that is 2–10 times larger than the volume of erupted material and that is consistent with geophysical estimates. Although intermittent gas transfer events can be inferred by the development of ²¹⁰Pb excess, there is no evidence at Rabaul for a direct link between eruptive style, gas flux and (²¹⁰Pb/²²⁶Ra).11 page(s

NMR Solution Structure of a Photoswitchable Apoptosis Activating Bak Peptide Bound to Bcl-x(L)

The Bcl-2 family of proteins includes the major regulators and effectors of the intrinsic apoptosis pathway. Cancers are frequently formed when activation of the apoptosis mechanism is compromised either by misregulated expression of prosurvival family members or, more frequently, by damage to the regulatory pathways that trigger intrinsic apoptosis. Short peptides derived from the pro-apoptotic members of the Bcl-2 family can activate mechanisms that ultimately lead to cell death. The recent development of photocontrolled peptides that are able to change their conformation and activity upon irradiation with an external light source has provided new tools to target cells for apoptosis induction with temporal and spatial control. Here, we report the first NMR solution structure of a photoswitchable peptide derived from the proapoptotic protein Bak in complex with the antiapoptotic protein Bcl-xL. This structure provides insight into the molecular mechanism, by which the increased affinity of such photopeptides compared to their native forms is achieved, and offers a rationale for the large differences in the binding affinities between the helical and nonhelical states

Robust optical autofocus system utilizing neural networks trained for extended range and time-course and automated multiwell plate imaging including single molecule localization microscopy.

We present a robust, long-range optical autofocus system for microscopy utilizing machine learning. This can be useful for experiments with long image data acquisition times that may be impacted by defocusing resulting from drift of components, e.g. due to changes in temperature or mechanical drift. It is also useful for automated slide scanning or multiwell plate imaging where the sample(s) to be imaged may not be in the same horizontal plane throughout the image data acquisition. To address the impact of (thermal or mechanical) fluctuations over time in the optical autofocus system itself, we utilise a convolutional neural network (CNN) that is trained over multiple days to account for such fluctuations. To address the trade-off between axial precision and range of the autofocus, we implement orthogonal optical readouts with separate CNN training data, thereby achieving an accuracy well within the 600 nm depth of field of our 1.3 numerical aperture objective lens over a defocus range of up to approximately +/-100 μm. We characterise the performance of this autofocus system and demonstrate its application to automated multiwell plate single molecule localisation microscopy. This article is protected by copyright. All rights reserved

NMR Solution Structure of a Photoswitchable Apoptosis Activating Bak Peptide Bound to Bcl-x_L

The Bcl-2 family of proteins includes the major regulators and effectors of the intrinsic apoptosis pathway. Cancers are frequently formed when activation of the apoptosis mechanism is compromised either by misregulated expression of prosurvival family members or, more frequently, by damage to the regulatory pathways that trigger intrinsic apoptosis. Short peptides derived from the pro-apoptotic members of the Bcl-2 family can activate mechanisms that ultimately lead to cell death. The recent development of photocontrolled peptides that are able to change their conformation and activity upon irradiation with an external light source has provided new tools to target cells for apoptosis induction with temporal and spatial control. Here, we report the first NMR solution structure of a photoswitchable peptide derived from the proapoptotic protein Bak in complex with the antiapoptotic protein Bcl-x_L. This structure provides insight into the molecular mechanism, by which the increased affinity of such photopeptides compared to their native forms is achieved, and offers a rationale for the large differences in the binding affinities between the helical and nonhelical states