ABSTRACT

During infection of human parvovirus B19 (B19V), one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter and is alternatively spliced and alternatively polyadenylated. Here, we identified a novel cis-acting sequence (5'-GUA AAG CUA CGG GAC GGU-3'), intronic splicing enhancer 3 (ISE3), which lies 72 nucleotides upstream of the second splice acceptor (A2-2) site of the second intron that defines the exon of the mRNA encoding the 11-kDa viral nonstructural protein. RNA binding motif protein 45 (RBM45) specifically binds to ISE3 with high affinity (equilibrium dissociation constant [K_D] = 33 nM) mediated by its RNA recognition domain and 2-homo-oligomer assembly domain (RRM2-HOA). Knockdown of RBM45 expression or ectopic overexpression of RRM2-HOA in human erythroid progenitor cells (EPCs) expanded ex vivo significantly decreased the level of viral mRNA spliced at the A2-2 acceptor but not that of the mRNA spliced at A2-1 that encodes VP2. Moreover, silent mutations of ISE3 in an infectious DNA of B19V significantly reduced 11-kDa expression. Notably, RBM45 also specifically interacts in vitro with ISE2, which shares the octanucleotide (GGGACGGU) with ISE3. Taken together, our results suggest that RBM45, through binding to both ISE2 and ISE3, is an essential host factor for maturation of 11-kDa-encoding mRNA.

IMPORTANCE Human parvovirus B19 (B19V) is a human pathogen that causes severe hematological disorders in immunocompromised individuals. B19V infection has a remarkable tropism with respect to human erythroid progenitor cells (EPCs) in human bone marrow and fetal liver. During B19V infection, only one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter of the viral genome and is alternatively spliced and alternatively polyadenylated, a process which plays a key role in expression of viral proteins. Our studies revealed that a cellular RNA binding protein, RBM45, binds to two intron splicing enhancers and is essential for the maturation of the small nonstructural protein 11-kDa-encoding mRNA. The 11-kDa protein plays an important role not only in B19V infection-induced apoptosis but also in viral DNA replication. Thus, the identification of the RBM45 protein and its cognate binding site in B19V pre-mRNA provides a novel target for antiviral development to combat B19V infection-caused severe hematological disorders.

Group II introns are mobile genetic elements that perform both self-splicing and intron mobility reactions. These ribozymes are comprised of a catalytic RNA core that binds to an intron-encoded protein (IEP) to form a ribonucleoprotein (RNP) complex. Splicing proceeds through two competing reactions: hydrolysis or branching. Group IIC intron ribozymes have a minimal RNA architecture, and splice almost exclusively through hydrolysis in ribozyme reactions. Addition of the IEP allows the splicing reaction to form branched lariat RNPs capable of intron mobility. Here we examine ribozyme splicing, IEP-dependent splicing, and mobility reactions of a group IIC intron from the thermophilic bacterium Thermoanerobacter italicus (Ta.it.I1). We show that Ta.it.I1 is highly active for ribozyme activity, forming linear hydrolytic intron products. Addition of purified IEP switches activity to the canonical lariat forming splicing reaction. We demonstrate that the Ta.it.I1 group IIC intron coordinates the progression of the forward splicing reaction through a –' interaction between intron domains II and VI. We further show that branched splicing is supported in the absence of the IEP when the –' interaction is mutated. We also investigated the regulation of the two steps of reverse splicing during intron mobility into DNA substrates. Using a fluorescent mobility assay that simultaneously visualizes all steps of intron integration into DNA, we show that completion of reverse splicing is tightly coupled to cDNA synthesis regardless of mutation of the –' interaction.

Thematic collection: This article is part of the Exploration 17 collection available at: https://www.lyellcollection.org/cc/exploration-17

Megakaryoblastic leukemia 1 (MKL1) is a coactivator of serum response factor and together they regulate transcription of actin cytoskeleton genes. MKL1 is associated with hematologic malignancies and immunodeficiency, but its role in B cells is unexplored. Here we examined B cells from monozygotic triplets with an intronic deletion in MKL1, two of whom had been previously treated for Hodgkin lymphoma (HL). To investigate MKL1 and B-cell responses in the pathogenesis of HL, we generated Epstein-Barr virus-transformed lymphoblastoid cell lines from the triplets and two controls. While cells from the patients with treated HL had a phenotype close to that of the healthy controls, cells from the undiagnosed triplet had increased MKL1 mRNA, increased MKL1 protein, and elevated expression of MKL1-dependent genes. This profile was associated with elevated actin content, increased cell spreading, decreased expression of CD11a integrin molecules, and delayed aggregation. Moreover, cells from the undiagnosed triplet proliferated faster, displayed a higher proportion of cells with hyperploidy, and formed large tumors in vivo. This phenotype was reversible by inhibiting MKL1 activity. Interestingly, cells from the triplet treated for HL in 1985 contained two subpopulations: one with high expression of CD11a that behaved like control cells and the other with low expression of CD11a that formed large tumors in vivo similar to cells from the undiagnosed triplet. This implies that pre-malignant cells had re-emerged a long time after treatment. Together, these data suggest that dysregulated MKL1 activity participates in B-cell transformation and the pathogenesis of HL.

Honouring real estate developers who are relentless in their pursuit to be socially and environmentally responsible.

The latest version of ANYbotics' four-legged robot can do useful real-world inspection tasks

The Danish company is announcing a powerful new member of its collaborative robots family

The remains of three slaves found in Mexico contain the earliest signs of the hepatitis B virus and yaws bacteria in the Americas, suggesting transatlantic slavery introduced these diseases

Stand-up comic and actor recreated his 2005 sketch to raise money for Comic Relief and Children in Need

The new standards don’t do much to compel the aviation industry to reduce emissions.

The group brings experts and policy makers together

The NRL is introducing a self-isolation program for its players, as it looks to continue the premiership amid the coronavirus pandemic.

In 2006, I launched the Drug Channels website to explore the economics of the U.S. reimbursement and distribution system for prescription pharmaceuticals. More than 30,000 professionals in the pharmaceutical and related industries rely on Drug Channels for timely analysis and provocative, fact-based insights.

Today, I am pleased to announce the launch of our new delivery platform: Drug Channels Video!

You can look forward to:

• Information and analysis via our new YouTube channel (Never fear. I’ll still be publishing articles on the website every week.)

• Livestream video webinars (Stay tuned next week for an announcement about our first two events.)

• Private, live virtual keynotes and remote presentations (Email me to schedule a safe social distancing event for your team.)

• Online video courses (Coming later in 2020)

Please watch my brief video introduction below. (Click here if you can’t see the video.)


I hope you and your families stay safe and healthy in these challenging times. I look forward to reconnecting with you in person soon.

"The Asset Forfeiture Program is critical to our efforts to reduce and deter criminal activity. Your use of civil and criminal forfeiture to seize and forfeit the assets of organized crime targets is essential for this fight."

"Now, there’s no question that we can be proud of the anti-violence work that’s already under way - and the progress that so many of you are leading - in big cities and small towns across the country. But, as this report clearly illustrates, we cannot yet be satisfied - and this is no time to become complacent," said Attorney General Holder.

It is a home telephone service which gives users unlimited local and nationwide calling in the US. For international calls, users will get the same rates used as the Google Voice service.

On this episode, a discussion about a new Brookings resource called Techstream, a publication site on brookings.edu that puts technologists and policymakers in conversation. Chris Meserole, a fellow in Foreign Policy and deputy director of the Artificial Intelligence and Emerging Technology Initiative, explains what Techstream is and some of the issues it covers. Also on…

On this episode, a discussion about a new Brookings resource called Techstream, a publication site on brookings.edu that puts technologists and policymakers in conversation. Chris Meserole, a fellow in Foreign Policy and deputy director of the Artificial Intelligence and Emerging Technology Initiative, explains what Techstream is and some of the issues it covers. Also on…

On this episode, a discussion about a new Brookings resource called Techstream, a publication site on brookings.edu that puts technologists and policymakers in conversation. Chris Meserole, a fellow in Foreign Policy and deputy director of the Artificial Intelligence and Emerging Technology Initiative, explains what Techstream is and some of the issues it covers. Also on…

This is what has been touted as the greenest company in America. Will they give vinyl a green sheen?

Many of Obama's rules never came into force, and many of Trump's will never make much difference.

My name is Daniel Wallach, and I am Executive Director of Greensburg GreenTown. My wife, Catherine Hart, is the Coordinator of

This Monopoly alternative is designed to act as a walkthrough of the concepts of a new economically and ecologically viable "Industrial Evolution" based on a renewable bioeconomy.

Industrial hemp farming could play a big role in providing economic stability in impoverished areas, creating jobs and businesses for veterans, and growing a sustainable and regenerative agriculture movement.

It's always about the car, but this Florida company is thinking about the rest of us.

Photo credit: Wikipedia/Creative Commons 24 of the top teachers in the U.S. have been chosen to go to the Galapagos Islands, with the Toyota International Teacher Program. The program is designed to engage a variety of conservation and education issues

It is better than a fixed standing desk? They make a good case for it.

Drivers used to get away with murder; new legislation is much stronger.

Help Treehugger founder Graham Hill design his compact, off grid, energy positive compound in Maui.

Every country everywhere should be doing this.

This is where we started.

Finally, fire departments are buying equipment designed for the city instead of designing the city to fit the equipment.

Few things make as much sense as eating a harmful species, while sparing the threatened ones.

It's going for every label from LEED to Living Building Challenge

Self-driving cars will likely be big autonomous mobile home theaters.

It runs the full width of the car. What could possibly be the problem?

It's a moveable feast of cheaper, cooler, greener electricity for building blockchains.

Now how about some incentives to get people out of cars?

Every company should have to do this– more useful information that consumers need.

The tiny house movement goes to the dogs with this $30,000 dream house.

Load up the Krug and the caviar and go for a spin.

A mix of modern and traditional design elements that are appropriate for the Northeast make these very interesting houses.

This is, whether we like it or not, the future of furniture design.

The LightCycle follows our circadian rhythms via your phone and GPS.

We all walk on it every day, but don't often think of it. It's underfoot, and, for many of us, out of mind. But just because you trod on it doesn't mean you shouldn't pay close attention to the materials that make