Precise gene models using long-read sequencing reveal a unique poly(A) signal in Giardia lamblia.

During pre-mRNA processing, the poly(A) signal is recognized by a protein complex that ensures precise cleavage and polyadenylation of the nascent transcript. The location of this cleavage event establishes the length and sequence of the 3' UTR of an mRNA, thus determining much of its post-transcriptional fate. Using long-read sequencing, we characterize the polyadenylation signal and related sequences surrounding Giardia lamblia cleavage sites for over 2600 genes. We find that G. lamblia uses an AGURAA poly(A) signal, which differs from the mammalian AAUAAA. We also describe how G. lamblia lacks common auxiliary elements found in other eukaryotes, along with the proteins that recognize them. Further, we identify 133 genes with evidence of alternative polyadenylation. These results suggest that despite pared-down cleavage and polyadenylation machinery, 3' end formation still appears to be an important regulatory step for gene expression in G. lamblia.

Posttranscriptional Regulation by Proteins and Noncoding RNAs.

Posttranscriptional regulation comprises those mechanisms occurring after the initial copy of the DNA sequence is transcribed into an intermediate RNA molecule (i.e., messenger RNA) until such a molecule is used as a template to generate a protein. A subset of these posttranscriptional regulatory mechanisms essentially are destined to process the immature mRNA toward its mature form, conferring the adequate mRNA stability, providing the means for pertinent introns excision, and controlling mRNA turnover rate and quality control check. An additional layer of complexity is added in certain cases, since discrete nucleotide modifications in the mature RNA molecule are added by RNA editing, a process that provides large mature mRNA diversity. Moreover, a number of posttranscriptional regulatory mechanisms occur in a cell- and tissue-specific manner, such as alternative splicing and noncoding RNA-mediated regulation. In this chapter, we will briefly summarize current state-of-the-art knowledge of general posttranscriptional mechanisms, while major emphases will be devoted to those tissue-specific posttranscriptional modifications that impact on cardiac development and congenital heart disease.

The end of the message: multiple protein-RNA interactions define the mRNA polyadenylation site.

The key RNA sequence elements and protein factors necessary for 3' processing of polyadenylated mRNA precursors are well known. Recent studies, however, have significantly reshaped current models for the protein-RNA interactions involved in poly(A) site recognition, painting a picture more complex than previously envisioned and also providing new insights into regulation of this important step in gene expression. Here we review the recent advances in this area and provide a perspective for future studies.

Reconstitution of CPSF active in polyadenylation: recognition of the polyadenylation signal by WDR33.

Cleavage and polyadenylation specificity factor (CPSF) is the central component of the 3' processing machinery for polyadenylated mRNAs in metazoans: CPSF recognizes the polyadenylation signal AAUAAA, providing sequence specificity in both pre-mRNA cleavage and polyadenylation, and catalyzes pre-mRNA cleavage. Here we show that of the seven polypeptides that have been proposed to constitute CPSF, only four (CPSF160, CPSF30, hFip1, and WDR33) are necessary and sufficient to reconstitute a CPSF subcomplex active in AAUAAA-dependent polyadenylation, whereas CPSF100, CPSF73, and symplekin are dispensable. WDR33 is required for binding of reconstituted CPSF to AAUAAA-containing RNA and can be specifically UV cross-linked to such RNAs, as can CPSF30. Transcriptome-wide identification of WDR33 targets by photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) showed that WDR33 binds in and very close to the AAUAAA signal in vivo with high specificity. Thus, our data indicate that the large CPSF subunit participating in recognition of the polyadenylation signal is WDR33 and not CPSF160, as suggested by previous studies.

Genome-wide identification and predictive modeling of polyadenylation sites in eukaryotes.

Polyadenylation [poly(A)] is a vital step in post-transcriptional processing of pre-mRNA. Alternative polyadenylation is a widespread mechanism of regulating gene expression in eukaryotes. Defining poly(A) sites contributes to the annotation of transcripts' ends and the study of gene regulatory mechanisms. Here, we survey methods for collecting poly(A) sites using high-throughput sequencing technologies and summarize the general processes for genome-wide poly(A) site identifications. We also compare the performances of various poly(A) site prediction models and discuss the relationship between poly(A) site identification from sequencing projects and predictive modeling. Moreover, we attempt to address some potential problems in current researches and propose future directions related to polyadenylation research.

PAS-seq 2: A fast and sensitive method for global profiling of polyadenylated RNAs.

Alternative polyadenylation (APA) is a widespread phenomenon in eukaryotes that contributes to regulating gene expression and generating proteomic diversity. APA plays critical roles in development and its mis-regulation has been implicated in a wide variety of human diseases, including cancer. To study APA on the transcriptome-wide level, numerous deep sequencing methods that capture 3' end of mRNAs have been developed in the past decade, but they generally require a large amount of hands-on time and/or high RNA input. Here, we introduce PAS-seq 2, a fast and sensitive method for global and quantitative profiling of polyadenylated RNAs. Compared to our original PAS-seq, this method takes less time and requires much lower total RNA input due to improvement in the reverse transcription process. PAS-seq 2 can be applied to both APA and differential gene expression analyses.

Using TIF-Seq2 to investigate association between 5´ and 3´mRNA ends.

The development of high-throughput technologies has revealed pervasive transcription in all genomes that have been investigated so far. This has uncovered a highly interleaved transcriptome organization involving thousands of overlapping coding and non-coding RNA isoforms that challenge our traditional definitions of genes and functional regions of the genome. In this chapter, we discuss the application of an improved Transcript Isoform Sequencing approach (TIF-Seq2) able to concurrently determine the start and end sites of individual RNA molecules. We exemplify its use for the investigation of the human transcriptome and show how it is especially well suited to discriminate between overlapping molecules and accurately define their boundaries.

Polyadenylation sites and their characteristics in the genome of channel catfish (Ictalurus punctatus) as revealed by using RNA-Seq data.

Polyadenylation plays important roles in gene expression regulation in eukaryotes, which typically involves cleavage and poly(A) tail addition at the polyadenylation site (PAS) of the pre-mature mRNA. Many eukaryotic genes contain more than one PASs, termed as alternative polyadenylation (APA). As a crucial post-transcriptional regulation, polyadenylation affects various aspects of RNA metabolism such as mRNA stability, translocation, and translation. However, polyadenylation has been rarely studied in teleosts. Here we conducted polyadenylation analysis in channel catfish, a commercially important aquaculture species around the world. Using RNA-Seq data, we identified 20,320 PASs which were classified into 14,500 clusters by merging adjacent PASs. Most of the PASs were found in 3' UTRs, followed by intron regions based on the annotation of channel catfish reference genome. No apparent difference in PAS distribution was observed between the sense and antisense strand of the channel catfish genome. The sequence analysis of nucleotide composition and motif around PASs yielded a highly similar profile among various organisms, suggesting the conservation and importance of polyadenylation in evolution. Using APA genes with more than two PASs, gene ontology enrichment revealed genes particularly involved in RNA binding. Reactome pathway analysis showed the enrichment of the innate immune system, especially neutrophil degranulation.

VAAPA: a web platform for visualization and analysis of alternative polyadenylation.

Polyadenylation [poly(A)] is an essential process during the maturation of most mRNAs in eukaryotes. Alternative polyadenylation (APA) as an important layer of gene expression regulation has been increasingly recognized in various species. Here, a web platform for visualization and analysis of alternative polyadenylation (VAAPA) was developed. This platform can visualize the distribution of poly(A) sites and poly(A) clusters of a gene or a section of a chromosome. It can also highlight genes with switched APA sites among different conditions. VAAPA is an easy-to-use web-based tool that provides functions of poly(A) site query, data uploading, downloading, and APA sites visualization. It was designed in a multi-tier architecture and developed based on Smart GWT (Google Web Toolkit) using Java as the development language. VAAPA will be a valuable addition to the community for the comprehensive study of APA, not only by making the high quality poly(A) site data more accessible, but also by providing users with numerous valuable functions for poly(A) site analysis and visualization.