Characterization of METTL16 as a cytoplasmic RNA binding protein

mRNA modification by N6-methyladenosine (m6A) is involved in many post-transcriptional regulation processes including mRNA stability, splicing and promotion of translation. Accordingly, the recently identified mRNA methylation complex containing METTL3, METTL14, and WTAP has been the subject of intense study. However, METTL16 (METT10D) has also been identified as an RNA m6A methyltransferase that can methylate both coding and noncoding RNAs, but its biological role remains unclear. While global studies have identified many potential RNA targets of METTL16, only a handful, including the long noncoding RNA MALAT1, the snRNA U6, as well as the mRNA MAT2A have been verified and/or studied to any great extent. In this study we identified/verified METTL16 targets by immunoprecipitation of both endogenous as well as exogenous FLAG-tagged protein. Interestingly, exogenously overexpressed METTL16 differed from the endogenous protein in its relative affinity for RNA targets which prompted us to investigate METTL16’s localization within the cell. Surprisingly, biochemical fractionation revealed that a majority of METTL16 protein resides in the cytoplasm of a number of cells. Furthermore, siRNA knockdown of METTL16 resulted in expression changes of a few mRNA targets suggesting that METTL16 may play a role in regulating gene expression. Thus, while METTL16 has been reported to be a nuclear protein, our findings suggest that METTL16 is also a cytoplasmic methyltransferase that may alter its RNA binding preferences depending on its cellular localization. Future studies will seek to confirm differences between cytoplasmic and nuclear RNA targets in addition to exploring the physiological role of METTL16 through long-term knockdown

MondoA-Mlx Heterodimers Are Candidate Sensors of Cellular Energy Status: Mitochondrial Localization and Direct Regulation of Glycolysis

Transcription factors can be sequestered at specific organelles and translocate to the nucleus in response to changes in organellar homeostasis. MondoA is a basic helix-loop-helix leucine zipper transcriptional activator similar to Myc in function. However, unlike Myc, MondoA and its binding partner Mlx localize to the cytoplasm, suggesting tight regulation of their nuclear function. We show here that endogenous MondoA and Mlx associate with mitochondria in primary skeletal muscle cells and erythroblast K562 cells. Interaction between MondoA and the mitochondria is salt and protease sensitive, demonstrating that it associates with the outer mitochondrial membrane by binding a protein partner. Further, endogenous MondoA shuttles between the mitochondria and the nucleus, suggesting that it communicates between these two organelles. When nuclear, MondoA activates transcription of a broad spectrum of metabolic genes, including those for the glycolytic enzymes lactate dehydrogenase A, hexokinase II, and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3. Regulation of these three targets is mediated by direct interaction with CACGTG sites in their promoters. Consistent with its regulation of glycolytic targets, MondoA is both necessary and sufficient for glycolysis. We propose that MondoA communicates information about the intracellular energy state between the mitochondria and the nucleus, resulting in transcriptional activation of glycolytic target genes

Characterization of METTL16 as a cytoplasmic RNA binding protein

mRNA modification by N6-methyladenosine (m6A) is involved in many post-transcriptionalregulation processes including mRNA stability, splicing and promotion of translation.Accordingly, the recently identified mRNA methylation complex containing METTL3,METTL14, and WTAP has been the subject of intense study. However, METTL16(METT10D) has also been identified as an RNA m6A methyltransferase that can methylateboth coding and noncoding RNAs, but its biological role remains unclear. While global studies have identified many potential RNA targets of METTL16, only a handful, including thelong noncoding RNA MALAT1, the snRNA U6, as well as the mRNA MAT2A have been verified and/or studied to any great extent. In this study we identified/verified METTL16 targetsby immunoprecipitation of both endogenous as well as exogenous FLAG-tagged protein.Interestingly, exogenously overexpressed METTL16 differed from the endogenous proteinin its relative affinity for RNA targets which prompted us to investigate METTL16"s localization within the cell. Surprisingly, biochemical fractionation revealed that a majority ofMETTL16 protein resides in the cytoplasm of a number of cells. Furthermore, siRNA knockdown of METTL16 resulted in expression changes of a few mRNA targets suggesting thatMETTL16 may play a role in regulating gene expression. Thus, while METTL16 has beenreported to be a nuclear protein, our findings suggest that METTL16 is also a cytoplasmicmethyltransferase that may alter its RNA binding preferences depending on its cellular localization. Future studies will seek to confirm differences between cytoplasmic and nuclearRNA targets in addition to exploring the physiological role of METTL16 through long-termknockdown

Self-reported sexually transmitted infection testing behaviour amongst incarcerated young male offenders: findings from a qualitative study

Introduction Sexually transmitted infections (STIs) are a major public health problem in the UK. Here we describe young men's self-reported STI testing behaviour, and explore why testing is and is not sought in two locales: the community and the Young Offender Institute (YOI). Methods In-depth interviews were conducted with 40 men, aged 16–20 years, whilst incarcerated in a Scottish YOI. The participants were purposively sampled using answers from a questionnaire administered to 67 inmates. Results The majority (n = 24) of those interviewed reported having undergone STI testing: eight in the community, 12 within the YOI, and four in both the community and the YOI. The extent to which they were worried about STIs and perceived themselves ‘at risk’ was important in understanding openness to testing. The convenience of testing within the YOI boosted the numbers seeking testing once incarcerated. Not getting tested in the YOI was due to not realising that it was available or not getting around to it rather than objecting to, or being embarrassed about, testing. Discussion Increasing awareness of the availability of STI testing within YOIs would be likely to result in higher uptake. An opt-out YOI STI screening programme would probably result in very high testing rates. Accessibility and convenience are key elements of testing procedures for this group, in both the YOI and community settings

The incidence of Trichomonas vaginalis

OBJECTIVES: Trichomoniasis (TV) is associated with an increased risk of acquisition of sexually transmitted diseases (STDs) and HIV. The purpose of this study is to evaluate factors associated with incidence TV among female STD clinic attendees in the USA. METHODS: Data were collected from women participating in a randomised controlled trial evaluating brief risk reduction counselling at the time of HIV testing to reduce sexually transmitted infections (STIs) incidence in STD clinics. Participants recruited from STD clinics underwent STI testing at baseline and 6-month follow-up. TV testing was performed using Nucleic Acid Amplification Test. RESULTS: 1704 participants completed study assessments. Prevalence of TV was 14.6%, chlamydia 8.6%, gonorrhoea 3.0%, herpes simplex virus 2 44.7% and HIV 0.4%. Cumulative 6-month incidence of TV was 7.5%. Almost 50% of the incident TV cases had TV at baseline and had received treatment. Factors associated with incidence of TV were having chlamydia, TV and HIV at baseline: TV relative risk (RR)=3.37 (95% CI 2.35 to 4.83, p<0.001); chlamydia RR=1.92 (95% CI 1.23 to 2.99, p=0.04); and HIV=1.59 (95% CI 1.01 to 2.50, p=0.047). CONCLUSIONS: Prevalent and incident TV is common among STD clinic attendees; and baseline TV is the main risk factor for incident TV, suggesting high rates of reinfection or treatment failures. This supports the importance of rescreening women after treatment for TV, evaluating current treatment regimens and programmes to ensure treatment of sexual partners. CLINICAL TRIAL NUMBER: NCT01154296