Regulation of alternative polyadenylation in the yeast Saccharomyces cerevisiae by histone H3K4 and H3K36 methyltransferases.

Adjusting DNA structure via epigenetic modifications, and altering polyadenylation (pA) sites at which precursor mRNA is cleaved and polyadenylated, allows cells to quickly respond to environmental stress. Since polyadenylation occurs cotranscriptionally, and specific patterns of nucleosome positioning and chromatin modifications correlate with pA site usage, epigenetic factors potentially affect alternative polyadenylation (APA). We report that the histone H3K4 methyltransferase Set1, and the histone H3K36 methyltransferase Set2, control choice of pA site in Saccharomyces cerevisiae, a powerful model for studying evolutionarily conserved eukaryotic processes. Deletion of SET1 or SET2 causes an increase in serine-2 phosphorylation within the C-terminal domain of RNA polymerase II (RNAP II) and in the recruitment of the cleavage/polyadenylation complex, both of which could cause the observed switch in pA site usage. Chemical inhibition of TOR signaling, which causes nutritional stress, results in Set1- and Set2- dependent APA. In addition, Set1 and Set2 decrease efficiency of using single pA sites, and control nucleosome occupancy around pA sites. Overall, our study suggests that the methyltransferases Set1 and Set2 regulate APA induced by nutritional stress, affect the RNAP II C-terminal domain phosphorylation at Ser2, and control recruitment of the 3 end processing machinery to the vicinity of pA sites.post-print1.432 K

Regulation of the Ysh1 endonuclease of the mRNA cleavage/polyadenylation complex by ubiquitinmediated degradation.

Mutation of the essential yeast protein Ipa1 has previously been demonstrated to cause defects in premRNA 3ʹ end processing and growth, but the mechanism underlying these defects was not clear. In this study, we show that the ipa1-1 mutation causes a striking depletion of Ysh1, the evolutionarily conserved endonuclease subunit of the 19-subunit mRNA Cleavage/Polyadenylation (C/P) complex, but does not decrease other C/P subunits. YSH1 overexpression rescues both the growth and 3ʹ end processing defects of the ipa1-1 mutant. YSH1 mRNA level is unchanged in ipa1-1 cells, and proteasome inactivation prevents Ysh1 loss and causes accumulation of ubiquitinated Ysh1. Ysh1 ubiquitination is mediated by the Ubc4 ubiquitin-conjugating enzyme and Mpe1, which in addition to its function in C/P, is also a RING ubiquitin ligase. In summary, Ipa1 affects mRNA processing by controlling the availability of the C/P endonuclease and may represent a regulatory mechanism that could be rapidly deployed to facilitate reprogramming of cellular responses.post-print4,60 M

A combined adjuvant approach primes robust germinal center responses and humoral immunity in non-human primates

Abstract Adjuvants and antigen delivery kinetics can profoundly influence B cell responses and should be critically considered in rational vaccine design, particularly for difficult neutralizing antibody targets such as human immunodeficiency virus (HIV). Antigen kinetics can change depending on the delivery method. To promote extended immunogen bioavailability and to present antigen in a multivalent form, native-HIV Env trimers are modified with short phosphoserine peptide linkers that promote tight binding to aluminum hydroxide (pSer:alum). Here we explore the use of a combined adjuvant approach that incorporates pSer:alum-mediated antigen delivery with potent adjuvants (SMNP, 3M-052) in an extensive head-to-head comparison study with conventional alum to assess germinal center (GC) and humoral immune responses. Priming with pSer:alum plus SMNP induces additive effects that enhance the magnitude and persistence of GCs, which correlate with better GC-TFH cell help. Autologous HIV-neutralizing antibody titers are improved in SMNP-immunized animals after two immunizations. Over 9 months after priming immunization of pSer:alum with either SMNP or 3M-052, robust Env-specific bone marrow plasma cells (BM BPC) are observed. Furthermore, pSer-modification of Env trimer reduce targeting towards immunodominant non-neutralizing epitopes. The study shows that a combined adjuvant approach can augment humoral immunity by modulating immunodominance and shows promise for clinical translation