Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability

Although correlations between RNA polymerase II (RNAPII) transcription stress, R-loops, and genome instability have been established, the mechanisms underlying these connections remain poorly understood. Here, we used a mutant version of the transcription elongation factor TFIIS (TFIISmut), aiming to specifically induce increased levels of RNAPII pausing, arrest, and/or backtracking in human cells. Indeed, TFIISmut expression results in slower elongation rates, relative depletion of polymerases from the end of genes, and increased levels of stopped RNAPII; it affects mRNA splicing and termination as well. Remarkably, TFIISmut expression also dramatically increases R-loops, which may form at the anterior end of backtracked RNAPII and trigger genome instability, including DNA strand breaks. These results shed light on the relationship between transcription stress and R-loops and suggest that different classes of R-loops may exist, potentially with distinct consequences for genome stability.Cancer Research UK FC001166UK Medical Research Council FC001166Wellcome Trust FC001166European Research Council 693327, ERC2014 AdG669898Ministerio de Economía y Competitividad BFU2013-42918-P, BFU2016-75058-

LARP7 suppresses P-TEFb activity to inhibit breast cancer progression and metastasis.

Transcriptional elongation by RNA polymerase (Pol) II is essential for gene expression during cell growth and differentiation. The positive transcription elongation factor b (P-TEFb) stimulates transcriptional elongation by phosphorylating Pol II and antagonizing negative elongation factors. A reservoir of P-TEFb is sequestered in the inactive 7SK snRNP where 7SK snRNA and the La-related protein LARP7 are required for the integrity of this complex. Here, we show that P-TEFb activity is important for the epithelial-mesenchymal transition (EMT) and breast cancer progression. Decreased levels of LARP7 and 7SK snRNA redistribute P-TEFb to the transcriptionally active super elongation complex, resulting in P-TEFb activation and increased transcription of EMT transcription factors, including Slug, FOXC2, ZEB2, and Twist1, to promote breast cancer EMT, invasion, and metastasis. Our data provide the first demonstration that the transcription elongation machinery plays a key role in promoting breast cancer progression by directly controlling the expression of upstream EMT regulators

Spt5 Cooperates with Human Immunodeficiency Virus Type 1 Tat by Preventing Premature RNA Release at Terminator Sequences

The human immunodeficiency virus type 1 (HIV-1) Tat protein activates transcription elongation by stimulating the Tat-activated kinase (TAK/p-TEFb), a protein kinase composed of CDK9 and its cyclin partner, cyclin T1. CDK9 is able to hyperphosphorylate the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase during elongation. In addition to TAK, the transcription elongation factor Spt5 is required for the efficient activation of transcriptional elongation by Tat. To study the role of Spt5 in HIV transcription in more detail, we have developed a three-stage Tat-dependent transcription assay that permits the isolation of active preinitiation complexes, early-stage elongation complexes, and Tat-activated elongation complexes. Spt5 is recruited in the transcription complex shortly after initiation. After recruitment of Tat during elongation through the transactivation response element RNA, CDK9 is activated and induces hyperphosphorylation of Spt5 in parallel to the hyperphosphorylation of the CTD of RNA polymerase II. However, immunodepletion experiments demonstrate that Spt5 is not required for Tat-dependent activation of the kinase. Chase experiments using the Spt5-depleted extracts demonstrate that Spt5 is not required for early elongation. However, Spt5 plays an important role in late elongation by preventing the premature dissociation of RNA from the transcription complex at terminator sequences and reducing the amount of polymerase pausing at arrest sites, including bent DNA sequences. This novel biochemical function of Spt5 is analogous to the function of NusG, an elongation factor found in Escherichia coli that enhances RNA polymerase stability on templates and shows sequence similarity to Spt5

DSK1, a novel kinesin-related protein from the diatom Cylindrotheca fusiformis that is involved in anaphase spindle elongation.

We have identified an 80-kD protein that is involved in mitotic spindle elongation in the diatom Cylindrotheca fusiformis. DSK1 (Diatom Spindle Kinesin 1) was isolated using a peptide antibody raised against a conserved region in the motor domain of the kinesin superfamily. By sequence homology, DSK1 belongs to the central motor family of kinesin-related proteins. Immunoblots using an antibody raised against a non-conserved region of DSK1 show that DSK1 is greatly enriched in mitotic spindle preparations. Anti-DSK1 stains in diatom central spindle with a bias toward the midzone, and staining is retained in the spindle midzone during spindle elongation in vitro. Furthermore, preincubation with anti-DSK1 blocks function in an in vitro spindle elongation assay. This inhibition of spindle elongation can be rescued by preincubating concurrently with the fusion protein against which anti-DSK1 was raised. We conclude that DSK1 is involved in spindle elongation and is likely to be responsible for pushing hal-spindles apart in the spindle midzone

In vitro reactivation of spindle elongation in fission yeast nuc2 mutant cells.

To investigate the mechanisms of spindle elongation and chromosome separation in the fission yeast Schizosaccharomyces pombe, we have developed an in vitro assay using a temperature-sensitive mutant strain, nuc2. At the restrictive temperature, nuc2 cells are arrested at a metaphase-like stage with short spindles and condensed chromosomes. After permeabilization of spheroplasts of the arrested cells, spindle elongation was reactivated by addition of ATP and neurotubulin both at the restrictive and the permissive temperatures, but chromosome separation was not. This suggests that the nuc2 cells are impaired in function at a stage before sister chromatid disjunction. Spindle elongation required both ATP and exogenous tubulin and was inhibited by adenylyl imidodiphosphate (AMPPNP) or vanadate. The ends of yeast half-spindle microtubules pulse-labeled with biotinylated tubulin moved past each other during spindle elongation and a gap formed between the original half-spindles. These results suggest that the primary mechanochemical event responsible for spindle elongation is the sliding apart of antiparallel microtubules of the two half-spindles

Polymer drift in a solvent by force acting on one polymer end

We investigate the effect of hydrodynamic interactions on the non-equilibrium drift dynamics of an ideal flexible polymer pulled by a constant force applied at one end of the polymer using the perturbation theory and the renormalization group method. For moderate force, if the polymer elongation is small, the hydrodynamic interactions are not screened and the velocity and the longitudinal elongation of the polymer are computed using the renormalization group method. Both the velocity and elongation are nonlinear functions of the driving force in this regime. For large elongation we found two regimes. For large force but finite chain length $L$ the hydrodynamic interactions are screened. For large chain lengths and a finite force the hydrodynamic interactions are only partially screened, which in three dimensions results in unusual logarithmic corrections to the velocity and the longitudinal elongation.Comment: 6 page

Actin-dependent cell elongation in teleost retinal rods: requirement for actin filament assembly.

Teleost retinal rods elongate when exposed to light. Elongation is mediated by a narrow necklike region called the myoid. In the cichlid Sarotherodon mossambicus, the rod inner segment (composed of the myoid with adjacent ellipsoid) increases in length from 12 micrometers in the dark to 41 micrometers in the light. Long light-adapted myoids contain longitudinally oriented microtubules and bundles of parallel 60-A filaments that we have identified as actin by their ability to bind myosin subfragment 1. In short dark-adapted myoids, only microtubules are recognizable. Colchicine experiments reveal that light-induced rod elongation can occur in the absence of myoid microtubules. Intraocular injections of colchicine at concentrations that disrupt virtually all rod myoid microtubules do not block rod elongation. However, rod elongation is blocked by intraocular injections of cytochalasin B or cytochalasin D. The hierarchy of effectiveness of these drugs is consistent with their effectiveness in inhibiting actin assembly and in disrupting other actin-dependent motile processes. On the basis of ultrastructural observations and the results of these inhibitor studies, we propose that the forces responsible for rod elongation are dependent not on microtubules but on actin filament assembly

Influence of Sheet Extensibility on Tearing Strength

This paper investigates the possibility that there is a relationship between the tearing properties and elongation properties of a sheet of paper. A lack of specific information in the literature and the advent of the in-plane tear method contributed to the need for work to be done in this area. Pulp was prepared according to TAPPI Standards and handsheets formed on a Noble And Wood sheet mold. After wet pressing, sheets were stretched with a hand made device to varying degrees, and dried in an oven in the stretched position. An Instron machine was used to determine the percent elongation, tensile energy absorption, and in-plane tear. An Elmendorf tear tester was used, also to determine tear. The results showed the in-plane tear to be very sensitive to elongation while the Elmendorf tear was not as sensitive. However, in both cases the tear did increase with an increase in sheet elongation. The reason for this occurring was due to more energy being dissipated throughout the sheet as the elongation increased

Regulation of transcription elongation in response to osmostress

Cells trigger massive changes in gene expression upon environmental fluctuations. The Hog1 stress-activated protein kinase (SAPK) is an important regulator of the transcriptional activation program that maximizes cell fitness when yeast cells are exposed to osmostress. Besides being associated with transcription factors bound at target promoters to stimulate transcriptional initiation, activated Hog1 behaves as a transcriptional elongation factor that is selective for stress-responsive genes. Here, we provide insights into how this signaling kinase functions in transcription elongation. Hog1 phosphorylates the Spt4 elongation factor at Thr42 and Ser43 and such phosphorylations are essential for the overall transcriptional response upon osmostress. The phosphorylation of Spt4 by Hog1 regulates RNA polymerase II processivity at stress-responsive genes, which is critical for cell survival under high osmostress conditions. Thus, the direct regulation of Spt4 upon environmental insults serves to stimulate RNA Pol II elongation efficiency.Ministerio de Economía y Competitividad BFU2015-64437-P, BFU2014-52125- REDT, BFU2014-51672-REDC BFU2014- 52333-P, BFU2016-77728-C3-1-P, BFU2013-48643-C3-1-PJunta de Andalucía P12-BIO1938MOGobierno de Cataluña 2014 SGR 59