Transcriptomic-wide discovery of direct and indirect HuR RNA targets in activated CD4+ T cells

Due to poor correlation between steady state mRNA levels and protein product, purely transcriptomic profiling methods may miss genes posttranscriptionally regulated by RNA binding proteins (RBPs) and microRNAs (miRNAs). RNA immunoprecipitation (RIP) methods developed to identify in vivo targets of RBPs have greatly elucidated those mRNAs which may be regulated via transcript stability and translation. The RBP HuR (ELAVL1) and family members are major stabilizers of mRNA. Many labs have identified HuR mRNA targets; however, many of these analyses have been performed in cell lines and oftentimes are not independent biological replicates. Little is known about how HuR target mRNAs behave in conditional knock-out models. In the present work, we performed HuR RIP-Seq and RNA-Seq to investigate HuR direct and indirect targets using a novel conditional knock-out model of HuR genetic ablation during CD4+ T activation and Th2 differentiation. Using independent biological replicates, we generated a high coverage RIP-Seq data set (>160 million reads) that was analyzed using bioinformatics methods specifically designed to find direct mRNA targets in RIP-Seq data. Simultaneously, another set of independent biological replicates were sequenced by RNA-Seq (>425 million reads) to identify indirect HuR targets. These direct and indirect targets were combined to determine canonical pathways in CD4+ T cell activation and differentiation for which HuR plays an important role. We show that HuR may regulate genes in multiple canonical pathways involved in T cell activation especially the CD28 family signaling pathway. These data provide insights into potential HuR-regulated genes during T cell activation and immune mechanisms

Leishmaniasis: An Evolving Public Health Concern in Thailand

Leishmaniasis is a parasitic disease caused by flagellated protozoa of the genus Leishmania. It is transmitted by the bite of an infected sandfly. The 3 main clinical forms of the disease are cutaneous leishmaniasis (CL), visceral leishmaniasis (VL) and mucocutaneous leishmaniasis (MCL). Prior to 1996, all leishmaniasis cases were infected during the visit to the endemic areas. Thereafter, autochthonous leishmaniasis cases have been reported in Thailand. From 1996 to the present, at least 21 cases of autochthonous leishmaniasis have been confirmed in Thailand. Leishmania siamensis, a novel species of Leishmania, was suspected of being the causative pathogens in some of those cases, However, the data supporting the existence of this new species is limited. Until recently, in-depth investigation using molecular characterization and isoenzyme analysis revealed that this suspected novel species consists of 2 different, but closely related strains: L. siamensis and L. martiniquensis. L. martiniquensis, a rare species firstly discovered on Martinique Island, is the cause of leishmaniasis in the majority of cases. Meanwhile, L. siamensis, a true novel species firstly and only reported from Thailand, was confirmed as the cause of leishmaniasis in two autochthonous cases. Two clinical forms (CL and VL) have been observed in both L. martiniquensis and L. siamensis infection. The DNA of L. martiniquensis was found in black rats, suggesting their role as a natural reservoir. The presence of L. martiniquensis DNA in two sandfly species (Sergentomyia gemmea and Sergentomyia barraudi) that are commonly found in affected areas may also suggest their role as potential vectors. Here, we update the status of leishmaniasis in Thailand and its emergence as a potential public health concern

Cell cycle RNA regulons coordinating early lymphocyte development

Lymphocytes undergo dynamic changes in gene expression as they develop from progenitor cells lacking antigen receptors, to mature cells that are prepared to mount immune responses. While transcription factors have established roles in lymphocyte development, they act in concert with post-transcriptional and post-translational regulators to determine the proteome. Furthermore, the post-transcriptional regulation of RNA regulons consisting of mRNAs whose protein products act cooperatively allows RNA binding proteins to exert their effects at multiple points in a pathway. Here, we review recent evidence demonstrating the importance of RNA binding proteins that control the cell cycle in lymphocyte development and discuss the implications for tumorigenesis. For further resources related to this article, please visit the WIREs website.</p

Posttranscriptional gene regulation of CD4+ T cell cytokine expression by the RNA binding protein HuR

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Posttranscriptional gene regulation by RNA-binding proteins such as HuR fine tunes gene expression in T cells leading to powerful effects on immune responses. HuR can stabilize target mRNAs and/or promote translation by interacting with their 3-UTR AUrich elements. We discovered that the interaction of HuR with the Il2ra (CD25) mRNA transcript is required for its optimal translation. Conditional HuR knockout (KO) in CD4+ T cells resulted in loss of IL-2 homeostasis and defects in Th2 cytokine production. In an allergic airway inflammatory model, OVA-challenged HuR KO mice had decreased lung cellular infiltration, eosinophilia and IL-13. These results demonstrate that HuR plays a pivotal role in maintaining normal IL-2 homeostasis, augmenting Th2 differentiation and promoting allergic airway inflammation

Posttranscriptional gene regulation of CD4+ T cell cytokine expression by the RNA binding protein HuR

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Posttranscriptional gene regulation by RNA-binding proteins such as HuR fine tunes gene expression in T cells leading to powerful effects on immune responses. HuR can stabilize target mRNAs and/or promote translation by interacting with their 3-UTR AUrich elements. We discovered that the interaction of HuR with the Il2ra (CD25) mRNA transcript is required for its optimal translation. Conditional HuR knockout (KO) in CD4+ T cells resulted in loss of IL-2 homeostasis and defects in Th2 cytokine production. In an allergic airway inflammatory model, OVA-challenged HuR KO mice had decreased lung cellular infiltration, eosinophilia and IL-13. These results demonstrate that HuR plays a pivotal role in maintaining normal IL-2 homeostasis, augmenting Th2 differentiation and promoting allergic airway inflammation.</jats:p

The RNA-binding protein HuR is required for normal homeostatic control of IL-2 expression in activated CD4 T cells (IRM6P.713)

Abstract Upon T cell activation approximately 50% of genes are regulated at the posttranscriptional level by RNA-binding proteins (RBPs). HuR is a stabilizing RBP which regulates mRNA via interactions with AU-rich elements (ARE) in mRNA 3’UTRs. HuR has been shown to be crucial for T cell development, thymic selection and egression. However, it is unclear how HuR regulates T cell differentiation and cytokine expression. To further investigate these questions, we generated a novel conditional HuR KO mouse, distal-Lck Cre ROSA HuRfl/fl which ablates HuR in mature T cells prior to activation. HuR KO T cells develop and egress from thymus normally; however, they display aberrant cytokine and receptor expression upon activation. Activated HuR KO T cells show striking increases in IL-2 (20-30 fold) and display an inability to turn off IL-2 expression, but significant decreases in IL-4, IL-5 and IL-13 with no change in IFN-γ. Over 90% of HuR KO CD4 cells still make IL-2 five days after activation but display impaired IL-4, IL-5 and IL-13 secretion. Increases in IL-2 in HuR KO cells are due to augmentation of IL-2 transcription and mRNA stability. Moreover, there are significant decreases in proliferation and CD25 (IL-2Rα) expression in HuR KO cells as a result of reductions in CD25 transcription. Thus, HuR is required for cytokine homeostasis in CD4 cells and may well function as a critical off-switch for IL-2 expression during T cell activation.</jats:p

An impact on medical student knowledge outcomes after replacing peer lectures with small group discussions

This article was migrated. The article was marked as recommended. Active learning has been proven as an effective teaching method that increases students' academic performance, satisfaction, and promotes life-long learning. A previous study showed that a peer lecture was time-consuming for both faculty members and students without any increase in knowledge outcome achievement of students when comparing to faculty lectures. Therefore, we replaced all peer teachings, taught in 2015, with faculty lectures and small group discussions in an organ-system part which focused mainly on applications of medical microbiology in 2016. The organ-system part was further divided into 3 sections according to type of organ systems. The knowledge outcome achievement was compared using MCQ scores. Peer lectures were mainly used as a teaching method only in 2015 (91.5%, 43 hours from a total of 47 hours) while none of them was used in 2016. On the other hand, SDG were mainly used as a teaching method only in 2016 (73.9%, 51 hours from a total of 69 hours). Students of 2016 had significantly higher average MCQ scores than those of 2015 only in the section 3 (80.8% as compared with 60.5%; p value &lt; 0.001). There was no significant difference in section 1, section 2, and overall MCQ scores. Apart from routine teaching preparation, there was no out-of-class preparation time for faculty lectures and SGD while peer teaching required at least 0.5-2 hours of extra out-of-class preparation time from at least 1 faculty member and 10-12 students per 1 hour of teaching. In conclusion, SGD provided equal or more knowledge outcome achievement of the student with less time-consuming than peer lecture. Therefore, at least in our teaching environment, SGD was proved to be a better option than a peer lecture for teaching applications of medical microbiology.</ns4:p

RNA-binding protein HuR controls IL-2 homeostasis in activated T cells by regulating CD25 expression (CCR3P.202)

Abstract Posttranscriptional gene regulation by RNA binding proteins (RBPs) and their microRNA (miRNAs) partners is known to affect 50% of genes during T cell activation. The RBP, HuR regulates mRNAs by binding to AU-rich elements (AREs) in transcript 3’UTRs. Many Th2 cytokines and IL-2 possess AREs. We hypothesized that HuR may play a critical role in T cell activation and differentiation via regulation of IL-2 signaling pathways. To test this, we generated a conditional HuR KO mouse, distal-lck Cre ROSA HuRfl/fl, in order to ablate HuR prior to T cell activation. HuR KO T cells developed and egressed from thymus normally. However, they displayed an inability to shut off IL-2 and maintain stable cell surface expression of IL-2Ra (CD25). HuR KO T cells had striking increases in IL-2 mRNA and protein but decreases in Th2 cytokines. They also had proliferation defects, decreased p-stat5 and reductions in CD25 and prdm-1 transcription. We determined that HuR protein binds to IL-2 and CD25 mRNAs by two independent methods. CD25 mRNA stability was unchanged despite increased IL-2. We quantitated polysomal loading in HuR KO T cells compared to controls and discovered significant reductions in CD25 mRNA recruitment to heavy polysomes. Therefore, HuR regulates T cell activation and IL-2 homeostasis by controlling CD25 expression and enabling translational efficiency. We conclude that HuR plays an indispensible role in normal IL-2 homeostasis and T cell activation.</jats:p