Modulation of exon skipping by high-affinity hnRNP A1-binding sites and by intron elements that repress splice site utilization.

The RNA-binding protein hnRNP A1 is a splicing regulator produced by exclusion of alternative exon 7B from the A1 pre-mRNA. Each intron flanking exon 7B contains a high-affinity A1-binding site. The A1-binding elements promote exon skipping in vivo, activate distal 5' splice site selection in vitro and improve the responsiveness of pre-mRNAs to increases in the concentration of A1. Whereas the glycine-rich C-terminal domain of A1 is not required for binding, it is essential to activate the distal 5' splice site. Because A1 complexes can interact simultaneously with two A1-binding sites, we propose that an interaction between bound A1 proteins facilitates the pairing of distant splice sites. Based on the distribution of putative A1-binding sites in various pre-mRNAs, an A1-mediated change in pre-mRNA conformation may help define the borders of mammalian introns. We also identify an intron element which represses the 3' splice site of exon 7B. The activity of this element is mediated by a factor distinct from A1. Our results suggest that exon 7B skipping results from the concerted action of several intron elements that modulate splice site recognition and pairing

Subtype-specific differences in transmission cluster dynamics of HIV-1 B and CRF01_AE in New South Wales, Australia

Introduction: The human immunodeficiency virus 1 (HIV-1) pandemic is characterized by numerous distinct sub-epidemics (clusters) that continually fuel local transmission. The aims of this study were to identify active growing clusters, to understand which factors most influence the transmission dynamics, how these vary between different subtypes and how this information might contribute to effective public health responses. Methods: We used HIV-1 genomic sequence data linked to demographic factors that accounted for approximately 70% of all new HIV-1 notifications in New South Wales (NSW). We assessed differences in transmission cluster dynamics between subtype B and circulating recombinant form 01_AE (CRF01_AE). Separate phylogenetic trees were estimated using 2919 subtype B and 473 CRF01_AE sequences sampled between 2004 and 2018 in combination with global sequence data and NSW-specific clades were classified as clusters, pairs or singletons. Significant differences in demographics between subtypes were assessed with Chi-Square statistics. Results: We identified 104 subtype B and 11 CRF01_AE growing clusters containing a maximum of 29 and 11 sequences for subtype B and CRF01_AE respectively. We observed a > 2-fold increase in the number of NSW-specific CRF01_AE clades over time. Subtype B clusters were associated with individuals reporting men who have sex with men (MSM) as their transmission risk factor, being born in Australia, and being diagnosed during the early stage of infection (p 1.5 sequences / 6-months) and which consisted of a majority of infections among MSM. We also found four active growing CRF01_AE clusters containing only infections among MSM. Finally, we found 47 subtype B and seven CRF01_AE clusters that contained a large gap in time (>1 year) between infections and may be indicative of intermediate transmissions via undiagnosed individuals. Conclusions: The large number of active and growing clusters among MSM are the driving force of the ongoing epidemic in NSW for subtype B and CRF01_AE