Tailer: A Pipeline for Sequencing-Based Analysis of Non-Polyadenylated RNA 3’ End Processing

Post-transcriptional trimming and tailing of RNA 3’ ends play key roles in the processing and quality control of non-coding RNAs (ncRNAs). However, bioinformatic tools to examine changes in the RNA 3’ “tailome” are sparse and not standardized. Here we present Tailer, a bioinformatic pipeline in two parts that allows for robust quantification and analysis of tail information from next generation sequencing experiments that preserve RNA 3’ end information. The first part of Tailer, Tailer-Processing, uses genome annotation or reference FASTA gene sequences to quantify RNA 3’ ends from SAM-formatted alignment files or FASTQ sequence read files produced from sequencing experiments. The second part, Tailer-Analysis, uses the output of Tailer-Processing to identify statistically significant RNA targets of trimming and tailing and create graphs for data exploration. We apply Tailer to RNA 3’ end sequencing experiments from three published studies and find that it accurately and reproducibly recapitulates key findings. Thus, Tailer should be a useful and easily accessible tool to globally investigate tailing dynamics of non-polyadenylated RNAs and conditions that perturb them.

ZFC3H1 prevents RNA trafficking into nuclear speckles through condensation

Controlling proper RNA pool for nuclear export is important for accurate gene expression. ZFC3H1 is a key controller that not only facilitates nuclear exosomal degradation, but also retains its bound polyadenylated RNAs in the nucleus upon exosome inactivation. However, how ZFC3H1 retains RNAs and how its roles in RNA retention and degradation are related remain largely unclear. Here, we found that upon degradation inhibition, ZFC3H1 forms nuclear condensates to prevent RNA trafficking to nuclear speckles (NSs) where many RNAs gain export competence. Systematic mapping of ZFC3H1 revealed that it utilizes distinct domains for condensation and RNA degradation. Interestingly, ZFC3H1 condensation activity is required for preventing RNA trafficking to NSs, but not for RNA degradation. Considering that no apparent ZFC3H1 condensates are formed in normal cells, our study suggests that nuclear RNA degradation and retention are two independent mechanisms with different preference for controlling proper export RNA pool—degradation is preferred in normal cells, and condensation retention is activated upon degradation inhibition.

Free circular introns with an unusual branchpoint in neuronal projections

The polarized structure of axons and dendrites in neuronal cells depends in part on RNA localization. Previous studies have looked at which polyadenylated RNAs are enriched in neuronal projections or at synapses, but less is known about the distribution of non-adenylated RNAs. By physically dissecting projections from cell bodies of primary rat hippocampal neurons and sequencing total RNA, we found an unexpected set of free circular introns with a non-canonical branchpoint enriched in neuronal projections. These introns appear to be tailless lariats that escape debranching. They lack ribosome occupancy, sequence conservation, and known localization signals, and their function, if any, is not known. Nonetheless, their enrichment in projections has important implications for our understanding of the mechanisms by which RNAs reach distal compartments of asymmetric cells.

The RNA Atlas, a single nucleotide resolution map of the human transcriptome

The human transcriptome consists of various RNA biotypes including multiple types of non-coding RNAs (ncRNAs). Current ncRNA compendia remain incomplete partially because they are almost exclusively derived from the interrogation of small- and polyadenylated RNAs. Here, we present a more comprehensive atlas of the human transcriptome that is derived from matching polyA-, total-, and small-RNA profiles of a heterogeneous collection of nearly 300 human tissues and cell lines. We report on thousands of novel RNA species across all major RNA biotypes, including a hitherto poorly-cataloged class of non-polyadenylated single-exon long non-coding RNAs. In addition, we exploit intron abundance estimates from total RNA-sequencing to test and verify functional regulation by novel non-coding RNAs. Our study represents a substantial expansion of the current catalogue of human ncRNAs and their regulatory interactions. All data, analyses, and results are available in the R2 web portal and serve as a basis to further explore RNA biology and function.

Free circular introns with an unusual branchpoint in neuronal projections

The polarized structure of axons and dendrites in neuronal cells depends in part on RNA localization. Previous studies have looked at which polyadenylated RNAs are enriched in neuronal projections or at synapses, but less is known about the distribution of non-adenylated RNAs. By physically dissecting projections from cell bodies of primary rat hippocampal neurons and sequencing total RNA, we found an unexpected set of free circular introns with a non-canonical branchpoint enriched in neuronal projections. These introns appear to be tailless lariats that escape debranching. They lack ribosome occupancy, sequence conservation, and known localization signals, and their function, if any, is not known. Nonetheless, their enrichment in projections has important implications for our understanding of the mechanisms by which RNAs reach distal compartments of asymmetric cells.

Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). To overcome these limitations, we have developed a novel protocol, Phenol Toluol extraction (PTex), that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a first global RNA-bound proteome of human HEK293 cells and Salmonella Typhimurium as a bacterial species.

LncRNAs: New Players in Apoptosis Control

The discovery that the mammalian genome is largely transcribed and that almost half of the polyadenylated RNAs is composed of noncoding RNAs has attracted the attention of the scientific community. Growing amount of data suggests that long noncoding RNAs (lncRNAs) are a new class of regulators involved not only in physiological processes, such as imprinting and differentiation, but also in cancer progression and neurodegeneration. Apoptosis is a well regulated type of programmed cell death necessary for correct organ development and tissue homeostasis. Indeed, cancer cells often show an inhibition of the apoptotic pathways and it is now emerging that overexpression or downregulation of different lncRNAs in specific types of tumors sensitize cancer cells to apoptotic stimuli. In this review we summarize the latest studies on lncRNAs and apoptosis with major attention to those performed in cancer cells and in healthy cells upon differentiation. We discuss the new perspectives of using lncRNAs as targets of anticancer drugs. Finally, considering that lncRNA levels have been reported to have a correlation with specific cancer types, we argue the possibility of using lncRNAs as tumor biomarkers.