RNA Recognition by the DNA End-Binding Ku Heterodimer

Most nucleic acid-binding proteins selectively bind either DNA or RNA, but not both nucleic acids. The Saccharomyces cerevisiae Ku heterodimer is unusual in that it has two very different biologically relevant binding modes: (1) Ku is a sequence-nonspecific double-stranded DNA end-binding protein with prominent roles in nonhomologous end-joining and telomeric capping, and (2) Ku associates with a specific stem–loop of TLC1, the RNA subunit of budding yeast telomerase, and is necessary for proper nuclear localization of this ribonucleoprotein enzyme. TLC1 RNA-binding and dsDNA-binding are mutually exclusive, so they may be mediated by the same site on Ku. Although dsDNA binding by Ku is well studied, much less is known about what features of an RNA hairpin enable specific recognition by Ku. To address this question, we localized the Ku-binding site of the TLC1 hairpin with single-nucleotide resolution using phosphorothioate footprinting, used chemical modification to identify an unpredicted motif within the hairpin secondary structure, and carried out mutagenesis of the stem–loop to ascertain the critical elements within the RNA that permit Ku binding. Finally, we provide evidence that the Ku-binding site is present in additional budding yeast telomerase RNAs and discuss the possibility that RNA binding is a conserved function of the Ku heterodimer

Identifying invasive species threats, pathways, and impacts to improve biosecurity

Managing invasive species with prevention and early-detection strategies can avert severe ecological and economic impacts. Horizon scanning, an evidence-based process combining risk screening and consensus building to identify threats, has become a valuable tool for prioritizing invasive species management and prevention. We assembled a working group of experts from academic, government, and nonprofit agencies and organizations, and conducted a multi-taxa horizon scan for Florida, USA, the first of its kind in North America. Our primary objectives were to identify high-risk species and their introduction pathways, to detail the magnitude and mechanism of potential impacts, and, more broadly, to demonstrate the utility of horizon scanning. As a means to facilitate future horizon scans, we document the process used to generate the list of taxa for screening. We evaluated 460 taxa for their potential to arrive, establish, and cause negative ecological and socioeconomic impacts, and identified 40 potential invaders, including alewife, zebra mussel, crab-eating macaque, and red swamp crayfish. Vertebrates and aquatic invertebrates posed the greatest invasion threat, over half of the high-risk taxa were omnivores, and there was high confidence in the scoring of high-risk taxa. Common arrival pathways were ballast water, biofouling of vessels, and escape from the pet/aquarium/horticulture trade. Competition, predation, and damage to agriculture/forestry/aquaculture were common impact mechanisms. We recommend full risk analysis for the high-risk taxa; increased surveillance at Florida's ports, state borders, and high-risk pathways; and periodic review and revision of the list. Few horizon scans detail the comprehensive methodology (including list-building), certainty estimates for all scoring categories and the final score, detailed pathways, and the magnitude and mechanism of impact. Providing this information can further inform prevention efforts and can be efficiently replicated in other regions. Moreover, harmonizing methodology can facilitate data sharing and enhance interpretation of results for stakeholders and the general public.</p

The 5′ Leader of the mRNA Encoding the Mouse Neurotrophin Receptor TrkB Contains Two Internal Ribosomal Entry Sites that Are Differentially Regulated

A single internal ribosomal entry site (IRES) in conjunction with IRES transactivating factors (ITAFs) is sufficient to recruit the translational machinery to a eukaryotic mRNA independent of the cap structure. However, we demonstrate that the mouse TrkB mRNA contains two independent IRESes. The mouse TrkB mRNA consists of one of two 5′ leaders (1428 nt and 448 nt), both of which include the common 3′ exon (Ex2, 344 nt). Dicistronic RNA transfections and in vitro translation of monocistronic RNA demonstrated that both full-length 5′ leaders, as well as Ex2, exhibit IRES activity indicating the IRES is located within Ex2. Additional analysis of the upstream sequences demonstrated that the first 260 nt of exon 1 (Ex1a) also contains an IRES. Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active. However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1). Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity in vitro and ex vivo, respectively. These results demonstrate that the two functionally independent IRESes within the mouse TrkB 5′ leader are differentially regulated, in part by PTB1

RNA structure-based ribosome recruitment: Lessons from the Dicistroviridae intergenic region IRESes

In eukaryotes, the canonical process of initiating protein synthesis on an mRNA depends on many large protein factors and the modified nucleotide cap on the 5′ end of the mRNA. However, certain RNA sequences can bypass the need for these proteins and cap, using an RNA structure-based mechanism called internal initiation of translation. These RNAs are called internal ribosome entry sites (IRESes), and the cap-independent initiation pathway they support is critical for successful infection by many viruses of medical and economic importance. In this review, we briefly describe and compare mechanistic and structural groups of viral IRES RNAs, focusing on those IRESes that are capable of direct ribosome recruitment using specific RNA structures. We then discuss in greater detail some recent advances in our understanding of the intergenic region IRESes of the Dicistroviridae, which use the most streamlined ribosome-recruitment mechanism yet discovered. By combining these findings with knowledge of canonical translation and the behavior of other IRESes, mechanistic models of this RNA structure-based process are emerging

The mouse TrkB 5′ leaders are able to initiate translation when cap-dependent translation is inhibited.

<p>A) A dual monocistronic construct containing the pGL3 multiple cloning site upstream of the <i>Renilla</i> luciferase and the pGL3 multiple cloning site, EMCV, or the TrkB 5′ leaders upstream of the <i>Photinus</i> luciferase gene was co-transfected into C6 cells with a construct encoding for a hypophosphorylated 4EBP construct or a null vector. The level of <i>Photinus</i> luciferase activity from each mRNA, when co-transfected with the null vector, was normalized to 100 percent. The <i>Renilla</i> and <i>Photinus</i> luciferase activity obtained in cells co-transfected with hypophosphorylated 4EBP is represented as a percentage of the activity obtained in cells co-transfected with the null plasmid. B) Monocistronic <i>Photinus</i> luciferase mRNA containing the β-globin or TrkB 5′ leaders was <i>in vitro</i> translated in rabbit reticulocyte lysate in the presence of increasing concentrations of cap analog. <i>Photinus</i> luciferase activity for each mRNA in the absence of cap analog was set to 100 percent.</p

The mouse TrkB 5′ leaders exhibit cryptic promoter activity.

<p>Promoterless dicistronic luciferase constructs containing the β-globin or the TrkB 5′ leaders were transfected into C6 cells. The P∶R ratio was normalized to that obtained from construct containing the β-globin 5′ leader.</p

PTB expression is required for IRES activity mediated by Ex2.

<p>A) Western blot analysis of PTB and GAPDH (as a loading control) from lysates obtained from differentiated and undifferentiated SH-SY5Y cells transfected with siRNA directed against PTB or mock transfected for 72 hours. B) Dicistronic RNA containing the two mouse TrkB IRESes were transfected into differentiated SH-SY5Y cells depleted of PTB1 by siRNA. The P∶R ratios were normalized to that from the mRNA containing the β-globin 5′ leader in control undifferentiated SH-SY5Y cells.</p

The mouse TrkB 5′ leaders exhibit IRES activity when expressed in dicistronic RNA constructs.

<p>Dicistronic luciferase mRNA containing the β-globin and the TrkB 5′ leaders were transfected into C6 cells. The P∶R ratio for each construct was normalized to that of the negative control, β-globin.</p

PTB increases IRES activity from the Ex2 IRES, but does not affect Ex1a IRES activity.

<p>A) Dicistronic luciferase mRNA containing the Ex1a, Ex2, or β-globin 5′ leader was <i>in vitro</i> translated in RRL that was either untreated or supplemented with 0.4 mg of PTB1. The change in the P∶R ratio for the samples when in the presence of PTB1 relative to the untreated sample are shown. B) The two mouse TrkB IRESes demonstrate differential regulation. SH-SY5Y cells treated with 2 mM retinoic acid or with DMSO (mock) for four days were transfected with dicistronic mRNA containing the β-globin, Ex2, or Ex1a 5′ leaders. The P∶R ratios were normalized to that from the mRNA containing the β-globin 5′ leader.</p