During chronic viral infection, virus-specific CD8(+) T cells become exhausted, exhibit poor effector function and lose memory potential. However, exhausted CD8(+) T cells can still contain viral replication in chronic infections, although the mechanism of this containment is largely unknown. Here we show that a subset of exhausted CD8(+) T cells expressing the chemokine receptor CXCR5 has a critical role in the control of viral replication in mice that were chronically infected with lymphocytic choriomeningitis virus (LCMV). These CXCR5(+) CD8(+) T cells were able to migrate into B-cell follicles, expressed lower levels of inhibitory receptors and exhibited more potent cytotoxicity than the CXCR5(-) [corrected] subset. Furthermore, we identified the Id2-E2A signalling axis as an important regulator of the generation of this subset. In patients with HIV, we also identified a virus-specific CXCR5(+) CD8(+) T-cell subset, and its number was inversely correlated with viral load. The CXCR5(+) subset showed greater therapeutic potential than the CXCR5(-) [corrected] subset when adoptively transferred to chronically infected mice, and exhibited synergistic reduction of viral load when combined with anti-PD-L1 treatment. This study defines a unique subset of exhausted CD8(+) T cells that has a pivotal role in the control of viral replication during chronic viral infection.

T cell activation is a well-established model for studying cellular responses to exogenous stimulation. Using strand-specific RNA-seq, we observed that intron retention is prevalent in polyadenylated transcripts in resting CD4+ T cells and is significantly reduced upon T cell activation. Several lines of evidence suggest that intron-retained transcripts are less stable than fully spliced transcripts. Strikingly, the decrease in intron retention (IR) levels correlate with the increase in steady-state mRNA levels. Further, the majority of the genes upregulated in activated T cells are accompanied by a significant reduction in IR. Of these 1583 genes, 185 genes are predominantly regulated at the IR level, and highly enriched in the proteasome pathway, which is essential for proper T cell proliferation and cytokine release. These observations were corroborated in both human and mouse CD4+ T cells. Our study revealed a novel post-transcriptional regulatory mechanism that may potentially contribute to coordinated and/or quick cellular responses to extracellular stimuli such as an acute infection.

Cellular senescence has been viewed as a tumor suppression mechanism and also as a contributor to individual aging. Widespread shortening of 3 ′ untranslated regions (3 ′ UTRs) in messenger RNAs (mRNAs) by alternative polyadenylation (APA) has recently been discovered in cancer cells. However, the role of APA in the process of cellular senescence remains elusive. Here, we found that hundreds of genes in senescent cells tended to use distal poly(A) (pA) sites, leading to a global lengthening of 3 ′ UTRs and reduced gene expression. Genes that harbor longer 3 ′ UTRs in senescent cells were enriched in senescence-related pathways. Rras2, a member of the Ras superfamily that participates in multiple signal transduction pathways, preferred longer 3 ′ UTR usage and exhibited decreased expression in senescent cells. Depletion of Rras2 promoted senescence, while rescue of Rras2 reversed senescence-associated phenotypes. Mechanistically, splicing factor TRA2B bound to a core "AGAA" motif located in the alternative 3 ′ UTR of Rras2, thereby reducing the RRAS2 protein level and causing senescence. Both proximal and distal poly(A) signals showed strong sequence conservation, highlighting the vital role of APA regulation during evolution. Our results revealed APA as a novel mechanism in regulating cellular senescence.

Human pluripotent stem cells (hPSCs) represent a unique opportunity for understanding the molecular mechanisms underlying complex traits and diseases. CRISPR/Cas9 is a powerful tool to introduce genetic mutations into the hPSCs for loss-of-function studies. Here, we developed an episomal vector-based CRISPR/Cas9 system, which we called epiCRISPR, for highly efficient gene knockout in hPSCs. The epiCRISPR system enables generation of up to 100% Insertion/Deletion (indel) rates. In addition, the epiCRISPR system enables efficient double-gene knockout and genomic deletion. To minimize off-target cleavage, we combined the episomal vector technology with double-nicking strategy and recent developed high fidelity Cas9. Thus the epiCRISPR system offers a highly efficient platform for genetic analysis in hPSCs.

Circular RNAs (circRNAs), a kind of covalently closed RNA molecule, were used to be considered a type of by-products of mis-splicing events and were discovered sporadically due to the technological limits in the early years. With the great technological progress such as high-throughput next-generation sequencing, numerous circRNAs have recently been detected in many species. CircRNAs were expressed in a spatio-temporally specific manner, suggesting their regulatory functional potentials were overlooked previously. Intriguingly, some circRNAs were indeed found with critical physiological functions in certain circumstances. CircRNAs have a more stable molecular structure that can resist to exoribonuclease comparing to those linear ones, and their molecular functions include microRNA sponge, regulatory roles in transcription, mRNA traps that compete with linear splicing, templates for translation and possibly other presently unknown roles. Here, we review the discovery and characterization of circRNAs, the origination and formation mechanism, the physiological functions and the molecular roles, along with the methods for detection of circRNAs. We further look into the future and propose key questions to be answered for these magical RNA molecules.