A temperature sensitive mutation in the CstF77 subunit of the polyadenylation complex reveals the critical function of mRNA 3' end formation for a robust heat stress response in plants

Background: Heat Shock Protein 101 (HSP101) in plants and orthologs in bacteria (Caseinolytic peptidase B, ClpB) and yeast (Hsp104) are essential for thermotolerance. To investigate molecular mechanisms of thermotolerance involving HSP101, we performed a suppressor screen in Arabidopsis thaliana of a semi-dominant, missense HSP101 allele, hot1-4 (A499T). Plants carrying the hot1-4 mutation are more heat-sensitive than an HSP101 null mutant (hot1-3), indicating the toxicity of hot1-4 allele. Results: We report that one suppressor (shot2, suppressor of hot1-4 2) has a temperature-sensitive, missense mutation (E170K) in the CstF77 (Cleavage stimulation factor 77) subunit of the polyadenylation complex, which is critical for 3' end maturation of pre-mRNA. RNA-Seq analysis of total RNA depleted of ribosomes reveals that heat treatment causes transcriptional readthrough events in shot2, specifically in highly heat-induced genes, including the toxic hot1-4 gene. In addition, failure of correct transcript processing leads to reduced accumulation of many HSP RNAs and proteins, suppressing heat sensitivity of the hot1-4 mutant, due to reduction of the toxic mutant HSP101 protein. Notably, the shot2 mutation makes plants more sensitive to heat stress in the HSP101 null (hot1-3) and wild-type backgrounds correlated with the reduced expression of other heat-inducible genes required for thermotolerance. Conclusions: Our study reveals the critical function of CstF77 for 3' end formation of mRNA during heat stress, as well as the dominant role of HSP101 in dictating the outcome of severe heat stress in plants.

Differential expression of cleavage stimulation factor subunit 2 in cancers of the breast.

Breast cancer affects women at relatively high frequency (1). We mined published microarray datasets (2, 3) to determine in an unbiased fashion and at the systems level genes most differentially expressed in the primary tumors of patients with breast cancer. We report here significant differential expression of the gene encoding the cleavage stimulation factor subunit 2, CSTF2, when comparing primary tumors of the breast to the tissue of origin, the normal breast. CSTF2 was also differentially expressed in the tumor cells of patients with triple negative breast cancer. CSTF2 mRNA was present at significantly higher quantities in tumors of the breast as compared to normal breast tissue. Analysis of human survival data revealed that expression of CSTF2 in primary tumors of the breast was correlated with overall survival in patients with basal subtype cancer. CSTF2 may be of relevance to initiation, maintenance or progression of cancers of the female breast.

mRNA Processing Factor CstF-50 and Ubiquitin Escort Factor p97 Are BRCA1/BARD1 Cofactors Involved in Chromatin Remodeling during the DNA Damage Response

ABSTRACT The cellular response to DNA damage is an intricate mechanism that involves the interplay among several pathways. In this study, we provide evidence of the roles of the polyadenylation factor cleavage stimulation factor 50 (CstF-50) and the ubiquitin (Ub) escort factor p97 as cofactors of BRCA1/BARD1 E3 Ub ligase, facilitating chromatin remodeling during the DNA damage response (DDR). CstF-50 and p97 formed complexes with BRCA1/BARD1, Ub, and some BRCA1/BARD1 substrates, such as RNA polymerase (RNAP) II and histones. Furthermore, CstF-50 and p97 had an additive effect on the activation of the ubiquitination of these BRCA1/BARD1 substrates during DDR. Importantly, as a result of these functional interactions, BRCA1/BARD1/CstF-50/p97 had a specific effect on the chromatin structure of genes that were differentially expressed. This study provides new insights into the roles of RNA processing, BRCA1/BARD1, the Ub pathway, and chromatin structure during DDR.

Polyadenylation proteins CstF-64 and τCstF-64 exhibit differential binding affinities for RNA polymers

CstF-64 (cleavage stimulation factor-64), a major regulatory protein of polyadenylation, is absent during male meiosis. Therefore a paralogous variant, τCstF-64 is expressed in male germ cells to maintain normal spermatogenesis. Based on sequence differences between τCstF-64 and CstF-64, and on the high incidence of alternative polyadenylation in testes, we hypothesized that the RBDs (RNA-binding domains) of τCstF-64 and CstF-64 have different affinities for RNA elements. We quantified Kd values of CstF-64 and τCstF-64 RBDs for various ribopolymers using an RNA cross-linking assay. The two RBDs had similar affinities for poly(G)18, poly(A)18 or poly(C)18, with affinity for poly(C)18 being the lowest. However, CstF-64 had a higher affinity for poly(U)18 than τCstF-64, whereas it had a lower affinity for poly(GU)9. Changing Pro-41 to a serine residue in the CstF-64 RBD did not affect its affinity for poly(U)18, but changes in amino acids downstream of the C-terminal α-helical region decreased affinity towards poly(U)18. Thus we show that the two CstF-64 paralogues differ in their affinities for specific RNA sequences, and that the region C-terminal to the RBD is important in RNA sequence recognition. This supports the hypothesis that τCstF-64 promotes germ-cell-specific patterns of polyadenylation by binding to different downstream sequence elements.

Polymorphism 10034C>T Is Located in a Functional Cleavage Stimulation Factor (CstF) Binding Site of the Fibrinogen Gamma Gene and Influences the γ′/γA mRNA Ratio.

Previously we found that haplotype 2 of the fibrinogen gamma gene (FGG-H2) is associated with an increased risk of deep venous thrombosis and with reduced fibrinogen γ′ levels, an alternatively spliced form of FGG, and reduced fibrinogen γ′/total fibrinogen ratios (Blood2005; 106(13): 4176–83). Two of the FGG-H2 tagging single nucleotide polymorphisms (SNPs), 9615C>T [rs2066864] and 10034C>T [rs2066865], are located in the region containing the two polyadenylation (pA) signals used in FGG pre-mRNA processing, namely pA1 and pA2, resulting in transcripts coding for γ′-chains and γA-chains, respectively. SNP 10034C>T is located in a Cleavage stimulation Factor (CstF) binding site at nucleotides 10030–10047 of FGG. CstF is a multi-subunit complex required for efficient cleavage and polyadenylation of pre-mRNAs. We hypothesized that 10034T, by strengthening this CstF binding site, favors use of pA2 (synthesis of γA-chains) at the expense of pA1-use (synthesis of γ′-chains). We transiently transfected FGG mini-gene constructs containing exon 9, intron 9 (containing pA1), exon 10 and the 3′ UTR (containing pA2) in liver-derived HepG2 cells and used quantitative real-time PCR to measure the mean relative pA-signal usage (pA1/pA2 ratio) of the different constructs. To study the effects of SNPs 9615C>T and 10034C>T on the relative use of pA1 and pA2, we made constructs carrying the different alleles of these SNPs. Compared to reference construct CC (9615C, 10034C; FGG-H1) of which the pA1/pA2 ratio was set at 100%, the ratio of construct TT (9615T, 10034T; FGG-H2) was 1.4-fold decreased (71.5%, p=0.015). To investigate whether the SNPs individually or collectively caused the reduction in the pA1/pA2 ratio, exchange constructs CT (9615C, 10034T) and TC (9615T, 10034C) were also tested. The ratio of construct CT was almost 1.2-fold decreased (85.3%, p=0.001), while the ratio of construct TC did not differ significantly from the reference construct (101.6%, p=0.890). These results indicate that in our in vitro system SNP 10034C>T, and not SNP 9615C>T, is the major contributor to the reduction of the pA1/pA2 ratio. We further investigated the functionality and importance of the CstF binding site, containing 10034C>T, with a construct in which nucleotides 10013–10056 containing the complete CstF binding site were deleted. Compared to the reference construct, in which the CstF site was still present, the pA1/pA2 ratio of the CstF deletion construct was 2.2-fold increased (222.3%, p<0.001), indicating that this site is involved in the regulation of pA2-usage. The functionality of the CstF site was further confirmed using several mutant constructs in which the CstF consensus was weakened or strengthened by introducing one or two point mutations. Weakening the CstF site resulted in a significant 1.5-fold increase of the pA1/pA2 ratio, while strengthening the site resulted in a significant 1.6-fold decrease of the ratio. From these results we conclude that 10034C>T is located in a functional CstF binding site and is involved in regulating the usage of the pA2-signal of FGG. We propose that the 10034C>T change is the functional variation in the FGG-H2 haplotype and is responsible for the reduction in the fibrinogen γ′/total fibrinogen ratio and the increased risk of deep venous thrombosis.