Transcripts of vaccinia virus postreplicative genes do not contain a 5’ methylguanosine cap

Vaccinia virus (VACV) is a prototypical poxvirus originally used for eradication of smallpox. Investigation into VACV mRNAs carried out almost half a century ago substantially contributed to the fundamental discovery of the 5’ mRNA cap, a hallmark of all eukaryotic and many viral mRNAs. VACV research also facilitated the identification and understanding of the general mechanism of 5’ mRNA cap synthesis. We characterized the VACV transcripts at the individual mRNA molecule level and found that vaccinia postreplicative mRNAs, containing nontemplated 5’ poly(A) leaders, surprisingly lack the 5’ cap structure in vivo. We also show that the lengths of the nontemplated leaders and the presence or absence of cap structures at the 5’ mRNA ends are controlled by the initiator sequence of the VACV postreplicative promoters.One Sentence SummaryThe promoter sequence determines the synthesis of the 5’ cap and poly(A) leaders in vaccinia virus postreplicative mRNAs.

Functional Cyclization of Eukaryotic mRNAs

The closed-loop model of eukaryotic translation states that mRNA is circularized by a chain of the cap-eIF4E-eIF4G-poly(A)-binding protein (PABP)-poly(A) interactions that brings 5′ and 3′ ends together. This circularization is thought to promote the engagement of terminating ribosomes to a new round of translation at the same mRNA molecule, thus enhancing protein synthesis. Despite the general acceptance and the elegance of the hypothesis, it has never been proved experimentally. Using continuous in situ monitoring of luciferase synthesis in a mammalian in vitro system, we show here that the rate of translation initiation at capped and polyadenylated reporter mRNAs increases after the time required for the first ribosomes to complete mRNA translation. Such acceleration strictly requires the presence of a poly(A)-tail and is abrogated by the addition of poly(A) RNA fragments or m7GpppG cap analog to the translation reaction. The optimal functional interaction of mRNA termini requires 5′ untranslated region (UTR) and 3′ UTR of moderate lengths and provides stronger acceleration, thus a longer poly(A)-tail. Besides, we revealed that the inhibitory effect of the dominant negative R362Q mutant of initiation factor eIF4A diminishes in the course of translation reaction, suggesting a relaxed requirement for ATP. Taken together, our results imply that, upon the functional looping of an mRNA, the recycled ribosomes can be recruited to the start codon of the same mRNA molecule in an eIF4A-independent fashion. This non-canonical closed-loop assisted reinitiation (CLAR) mode provides efficient translation of the functionally circularized mRNAs.

Novel Library Method for High-Throughput Full-Length Sequencing of the Immune Repertoire from Unseparated B-Cells with Single-Cell Resolutionution

High-throughput DNA sequencing of the adaptive immune receptor repertoire is a relatively new and fast growing technology used to study the immune response in health and disease. In B and T cell lymphoproliferative disorders, antigen receptor sequencing can be used to study clonal diversity and evolution of the disease in treatment free condition and in response to treatment. Furthermore, it can be used for the detection of minimal residual disease (MRD), providing information on the relationship between the presence and number of pre-treatment clone(s) and their relationship and responsibility for a subsequent relapse. The characteristics and quality of the data generated by high-throughput DNA sequencing of immune receptor signatures are the results of three major components: library preparation, sequencing platform, and software tools. For both the library and software, there are no standard protocols and tools. Indeed, new approaches are continually being developed to accommodate new sequencing platform features and shortcomings, such as errors and read length restrictions. Two major technical challenges are: procuring an unbiased repertoire library that for B lymphocytes obtains and retains the full length IGHV-D-J along with (sub)isotype information, and resolving data to a single cell level, crucial for detection of MRD and rare clonal variants existing in the early phase of the disease, which might emerge and be involved in future relapse or progression. We describe here a library preparation method for use with the Illumina MiSeq platform that results in an exhaustive full-length repertoire where virtually every B cell is sequenced, thereby maximizing the likelihood of identifying and quantifying the “real” IGHV-D-J repertoire of the sample analyzed. The method also allows the detection of very infrequent rearrangements and maintains IG sub-isotype information without compromising data quality. From 0.5 - 1 million human B cells can be sequenced in a single MiSeq 2x300 run with this approach. Key aspects of the technique are: 1) start from a well defined number of B lymphocytes 2) avoid V-gene specific PCR amplification and genetic material dilution in the pre-amplification phases 3) the specific depth of sequencing should depend on the starting B (or T) cell subset (i.e. na•ve, memory or plasma cell), and should be proportional to the number of starting cells. High quality sub-isotype information can be obtained with a second round of sequencing of shorter read length, e.g., with the Illumina 2x150 platform. We used 58 different CLL clones with known IGH sequence mixed all together with polyclonal B cell from a donor PBMC (Figure 1). The mixed lysate is used to test the ability to detect the different clones. The following describes how the absence of genetic material dilution in the pre-amplification phases impact on the ability to obtain a comprehensive repertoire. These are crucial in MRD detection, since diluting the genetic material (RNA and/or cDNA) prior PCR amplification compromises the ability to accurately and consistently detect the clonal variants, reducing the de facto sensitivity and reproducibility of the analysis. As a final example of the method's utility, we also demonstrate how different chronic lymphocytic leukemia clones present considerable variability in IG mRNA expression level that correlate with the number of unique mRNA molecule sequenced (Figure 3), which, if using a method with sub-optimal efficiency, could lead to a reduced clone-specific ability of detection by PCR based techniques. Figure 1. Figure 1. Figure 2. Each dilution is performed in replicates. The cDNA is obtained from all the RNA extracted from the starting cells. Each slice represents a different CLL, and each slice size is the frequency for which it is detected. A comprehensive detection of each CLL is dependent to the absence of genetic material dilution. Figure 2. Each dilution is performed in replicates. The cDNA is obtained from all the RNA extracted from the starting cells. Each slice represents a different CLL, and each slice size is the frequency for which it is detected. A comprehensive detection of each CLL is dependent to the absence of genetic material dilution. Figure 3. qPCR IgH expression correlate with the number of unique mRNA molecule sequenced. Figure 3. qPCR IgH expression correlate with the number of unique mRNA molecule sequenced. Disclosures No relevant conflicts of interest to declare.

A model for competition for ribosomes in the cell

A single mammalian cell includes an order of 10 4 –10 5 mRNA molecules and as many as 10 5 –10 6 ribosomes. Large-scale simultaneous mRNA translation induces correlations between the mRNA molecules, as they all compete for the finite pool of available ribosomes. This has important implications for the cell's functioning and evolution. Developing a better understanding of the intricate correlations between these simultaneous processes, rather than focusing on the translation of a single isolated transcript, should help in gaining a better understanding of mRNA translation regulation and the way elongation rates affect organismal fitness. A model of simultaneous translation is specifically important when dealing with highly expressed genes, as these consume more resources. In addition, such a model can lead to more accurate predictions that are needed in the interconnection of translational modules in synthetic biology. We develop and analyse a general dynamical model for large-scale simultaneous mRNA translation and competition for ribosomes. This is based on combining several ribosome flow models (RFMs) interconnected via a pool of free ribosomes. We use this model to explore the interactions between the various mRNA molecules and ribosomes at steady state. We show that the compound system always converges to a steady state and that it always entrains or phase locks to periodically time-varying transition rates in any of the mRNA molecules. We then study the effect of changing the transition rates in one mRNA molecule on the steady-state translation rates of the other mRNAs that results from the competition for ribosomes. We show that increasing any of the codon translation rates in a specific mRNA molecule yields a local effect, an increase in the translation rate of this mRNA, and also a global effect, the translation rates in the other mRNA molecules all increase or all decrease. These results suggest that the effect of codon decoding rates of endogenous and heterologous mRNAs on protein production is more complicated than previously thought. In addition, we show that increasing the length of an mRNA molecule decreases the production rate of all the mRNAs.

Combined Use of MS2 and PP7 Coat Fusions Shows that TIA-1 Dominates hnRNP A1 for K-SAM Exon Splicing Control

Splicing of the FGFR2 K-SAM exon is repressed by hnRNP A1 bound to the exon and activated by TIA-1 bound to the downstream intron. Both proteins are expressed similarly by cells whether they splice the exon or not, so it is important to know which one is dominant. To answer this question, we used bacteriophage PP7 and bacteriophage MS2 coat fusions to tether hnRNP A1 and TIA-1 to distinct sites on the same pre-mRNA molecule. hnRNP A1 fused to one coat protein was tethered to a K-SAM exon containing the corresponding coat protein's binding site. TIA-1 fused to the other coat protein was tethered to the downstream intron containing that coat protein's binding site. This led to efficient K-SAM exon splicing. Our results show that TIA-1 is dominant for K-SAM exon splicing control and validate the combined use of PP7 and MS2 coat proteins for studying posttranscriptional events.