“Synergistic assembly of human pre-spliceosomes across introns and exons”

In human genes, protein-coding regions alternate with non-coding “introns” that must be snipped out of the RNA transcript before it is used to produce a protein. The snipping is done by the spliceosome, a complex molecular machine that must assemble anew on each intron it removes.  The spliceosome must cut out exactly the segments of the messenger molecule that require removal, no more and no less, since inaccurate intron removal can produce a messenger molecule that is non-functional or that causes disease.

Implications of synergistic U2 recruitment for the mechanism of exon and intron recognition.To help understand how multiple introns are quickly and accurately removed, postdoctoral fellow Joerg Braun developed a light microscopy method by which for the first time we can observe the coordinated processes by which human spliceosomes recognize and assemble around the segments of single messenger RNA molecules.  As the eLife digest puts it: “The experiments show that spliceosomes working on different introns in the same pre-mRNA actually help each other out. As one assembles, this helps the spliceosome that processes the neighboring intron to get built. In particular, the U1snRNPs [a spliceosome sub-assembly] processing nearby introns collaborate to promote the assembly and activity of the spliceosomes. This teamwork is likely important to guarantee that multiple introns are cut out quickly and accurately.”

Synergistic assembly of human pre-spliceosomes across introns and exons.
Joerg E. Braun, Larry J. Friedman, Jeff Gelles, and Melissa J. Moore.
eLife (2018) 7:e37751.
New plasmids reported in this article can be obtained from Addgene.
Computer software used in this research can be obtained from GitHub.

“Mechanism and timing of Mcm2-7 ring closure during DNA replication origin licensing”

Mcm2-7 is a ring-shaped DNA helicase that plays an essential role in DNA repliction in eukaryotic cells.  Two of the helicase molecules must encircle the double-stranded DNA at a replication origin, establishing a loaded, anti-parallel double-ring complex able to start replication at the appropriate cell cycle stage.  In this study, Simina Ticau together with collaborators from Steve Bell’s lab (MIT), Jeff Gelles’ lab (Brandeis), and New England BioLabs used wild-type and mutant helicases in single-molecule colocalization (“CoSMoS”) and single-molecule fluorescence resonance energy transfer (smFRET) experiments to identify the mechanisms by which regulatory factors and nucleotide hydrolysis control ring opening and coordinate loading. This work reveals the molecular processes that serve to prevent catastrophic genome damage due to incorrect or mistimed assembly of the replicative machinery.

Mechanism and timing of Mcm2-7 ring closure during DNA replication origin licensing
Simina Ticau, Larry J Friedman, Kanokwan Champasa, Ivan R Corrêa Jr, Jeff Gelles, Stephen P Bell
Nat. Struct. Molec. Biol. (2017) 24: 309–315.

Video: Radcliffe Institute for Advanced Studies lecture

In February, Jeff gave a public lecture on “Seeing the Birth of an RNA Molecule” at the Radcliffe Institute for Advanced Studies.  This talk, intended for a scholarly audience consisting of both scientists and non-scientists, used single-molecule studies of transcription as examples of how visualization of molecular behavior has led to new insight into the mechanisms of fundamental molecular processes in biology.

Dynamics of GreB-RNA polymerase interaction

In living cells, messenger RNAs are not manufactured by RNA polymerases (RNAPs) functioning alone.  Instead, RNA synthesis is carried out collectively by RNAP together with accessory proteins that associate with the RNAP-containing transcription elongation complex and modulate its activity.  In this paper, Larry Tetone, Larry Friedman, and Melissa Osborne, along with their collaborators from the Gelles and Landick labs, used multi-wavelength single-molecule fluorescence methods to for the first time directly observe the dynamic binding and dissociation of an accessory protein with an RNAP during active transcript elongation.  The protein, GreB, is important for transcript proofreading in E. coli and other bacteria and is a functional analog of the TFIIS protein in eaukaryotes.  “Unexpectedly,” the authors report, “GreB was not selectively recruited to RNAPs requiring its transcript proofreading function. Instead, GreB transiently bound to multiple types of complexes, eventually finding via random search RNAPs that require its activity. The observations suggest a paradigm by which a regulator can act while minimizing obstruction of a binding site that must be shared with other proteins.”

Dynamics of GreB-RNA polymerase interaction allow a proofreading accessory protein to patrol for transcription complexes needing rescue
Larry E. Tetone, Larry J. Friedman, Melisa L. Osborne, Harini Ravi, Scotty Kyzer, Sarah K. Stumper, Rachel A. Mooney, Robert Landick, and Jeff Gelles
PNAS (2017) 114:E1081-E1090.

New plasmids reported in this article can be obtained from Addgene

“The dimerization equilibrium of a ClC Cl−/H+ antiporter in lipid bilayers”

The physical forces that drive oligomerization of soluble proteins are well understood and have been extensively studied.  For proteins with transmembrane domains — transport enzymes, for example — oligomerization is often essential for function but its physical basis is less clear.  In this project,  Janice Robertson devised a new method based on liposome extrusion and single-molecule fluorescence photobleaching analysis to accurately measure the dimer association free energy of a ClC-type chloride ion/hydrogen ion antiporter.  (Janice started this work when she was a postdoc in Chris Miller’s lab at Brandeis and later completed the project in her own  lab at the University of Iowa.)  The study reveals that ClC-ec1 “is one of the strongest membrane protein complexes measured so far, and introduces it as new type of dimerization model to investigate the physical forces that drive membrane protein association in membranes.”

The dimerization equilibrium of a ClC Cl−/H+ antiporter in lipid bilayers
Rahul Chadda, Venkatramanan Krishnamani, Kacey Mersch, Jason Wong, Marley Brimberry, Ankita Chadda, Ludmila Kolmakova-Partensky, Larry J Friedman, Jeff Gelles, and Janice L Robertson
eLife (2016) 5:e17438

Timothy Harden, Ph.D.

Congratulations! to Tim Harden, who successfully defended his Ph.D. dissertation in Physics with an additional specialization in Quantitative Biology.  Tim was jointly advised by Jeff Gelles and Jane Kondev.  He is now a Postdoctoral Fellow in Angela DePace’s lab at Harvard Medical School.

Jeff, Tim, Jane

L-to-R: Jeff, Tim, Jane

“Single molecule analysis reveals reversible and irreversible steps during spliceosome activation”

“The spliceosome is a complex molecular machine, composed of small nuclear ribonucleoproteins (snRNPs) and accessory proteins, that excises introns from precursor messenger RNAs (pre-mRNAs).  After assembly, the spliceosome is activated for catalysis by rearrangement of subunits to form an active site.” This study used multi-wavelength single-molecule fluorescence (“CoSMoS”) techniques to elucidate the mechanism of budding yeast spliceosome activation.  Activation turns out to be unexpectedly dynamic and variable: some spliceosomes  take multiple attempts to activate and the pathway contains both reversible and irreversible steps.  Strikingly, ATP powers both steps that drive the process forward toward splicing and well as reverse steps that diassemble intermediates to allow subsequent re-attempts at activation. These findings give new insight into how the efficiency and fidelity of pre-mRNA splicing is maintained.Predominant spliceosome activation pathway

The scientific project in this paper was initiated by Aaron Hoskins during his postdoctoral work in Melissa Moore’s and Jeff Gelles’ labs, but it was brought to fruition by Aaron and Margaret Rodgers working in Aaron’s lab at Univ. Wisconsin, Madison.

Single molecule analysis reveals reversible and irreversible steps during spliceosome activation
Aaron A. Hoskins Margaret L. Rodgers , Larry J. Friedman , Jeff Gelles , Melissa J. Moore
eLife (2016) 5:e14166

Radcliffe Fellow

During Jeff’s sabbatical in 2016-17, he will be a Fellow at the Radcliffe Institute for Advanced Study at Harvard University, working on a project to study eukaryotic mRNA synthesis and matuaration mechanisms using single-molecule fluorescence methods.  In addition to this new project he will also be spending time each week at Brandeis working with students and other scientists on the lab’s ongoing projects including those supported by NIH and the Mathers Foundation.  (The lab will be accepting Ph.D. students for rotation projects during 2016-17.)