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Non‐mRNA 3′ end formation: how the other half lives

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The release of nascent RNA from transcribing RNA polymerase complexes is required for all further functions carried out by RNA molecules. The elements and processing machinery involved in 3′ end formation therefore represent key determinants in the biogenesis and accumulation of cellular RNA. While these factors have been well‐characterized for messenger RNA, recent work has elucidated analogous pathways for the 3′ end formation of other important cellular RNA. Here, we discuss four specific cases of non‐mRNA 3′ end formation—metazoan small nuclear RNA, Saccharomyces cerevisiae small nuclear RNA, Schizosaccharomyces pombe telomerase RNA, and the mammalian MALAT1 large noncoding RNA—as models of alternative mechanisms to generate RNA 3′ ends. Comparison of these disparate processing pathways reveals an emerging theme of evolutionary ingenuity. In some instances, evidence for the creation of a dedicated processing complex exists; while in others, components are utilized from the existing RNA processing machinery and modified to custom fit the unique needs of the RNA substrate. Regardless of the details of how non‐mRNA 3′ ends are formed, the lengths to which biological systems will go to release nascent transcripts from their DNA templates are fundamental for cell survival.WIREs RNA 2013, 4:491–506. doi: 10.1002/wrna.1181 This article is categorized under: RNA Processing > 3' End Processing RNA Processing > Processing of Small RNAs

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Phylogenetic distribution of the integrator complex subunits. Each column represents a subunit of the Integrator complex. Int13 and Int14 correspond to the recently characterized subunits Asunder and CG4785, respectively. For each subunit, a search was performed against the corresponding genome(s) using Blastp with default parameters. The shading of each cell represents the level of identity between the human sequence and the considered organism(s). A white cell indicates that the search failed to return a sequence with significant homology. When a homolog is identified, it was verified that a reciprocal Blastp search using the identified subunit against the Metazoan taxon returned the corresponding integrator subunit as the most significant hit. Asterisks indicate that a significant homology was detected only on a portion of the protein sequence.
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MALAT1 noncoding RNA processing. (a) Schematic of genomic organization of MALAT1 and NEAT1 in human and mouse. (b) Schematic of the pathway of RNA processing events that lead to the generation of the MALAT1 mature transcript. The primary MALAT1 transcript contains a polyadenylation sequence (PAS) as well as a cloverleaf structure, which is a substrate for RNAseP. The downstream product is further processed by RNAseZ and the CCA‐adding enzyme preceding its export into the cytoplasm. The upstream MALAT1 transcript resulting from RNAseP digestion forms a triple helix at its 3′ end and is then localized to nuclear speckles.
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Schizosaccharomyces pombe telomerase RNA slicing. The primary TER1 transcript is produced by cleavage and polyadenylation and contains an intron near its 3′ end. Association of the Sm complex near the 5′ splice site promotes the first step of splicing; however, the second step of splicing does not occur. The high affinity of the U2snRNP for the branchpoint leads to a splicing discard pathway through the involvement of Prp43. The association of the Sm complex stimulates the hypermethylation of the TER1 5′ cap by Tgs1 and the complex is ultimately replaced with the Lsm complex, which both protects the 3′ terminus of the TER1 transcript and facilitates recruitment of the Trt1 protein component of the telomerase RNP.
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Mechanism of Nrd1/Nab3/Sen1‐dependent termination. The Nrd1/Nab3 hetereodimer recognizes the carboxy‐terminal domain (CTD) of Rpb1 that is phosphorylated at serine 5 within the RNA polymerase II (RNAPII) complex. Nrd1 contains the CTD interaction domain that recognizes this specific mark. In the absence of Rnt1, Nrd1, and Nab3 binding sites are exposed in the nascent snRNA transcript, which then facilitates termination by the Sen1 helicase. The termination product produces a free 3′ OH that is used as a substrate for the exosome or other 3′ to 5′ exonucleases.
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Schematic of Saccharomyces cerevisiae U4 and U5 snRNA 3′ end formation pathways. (a) U4 snRNA utilizes both Rnt1‐dependent and independent pathways to generate its 3′ end. The Rnt1‐dependent is related to U1 snRNA where the Rnt1 cleavage product acts as a substrate for the exosome and/or Rex2. The Rnt1‐independent pathway uses a termination site followed by exosome and/or Rex2 trimming. (b) The U5 snRNA utilizes the most complex Rnt1‐dependent and independent pathways. Cleavage by Rnt1 at two alternative sites (L or S) leads to the exosome/Rex2‐mediated trimming to either the U5L or U5S products. In the Rnt1‐independent pathway, a downstream termination element is used and the long U5 precursor is processed to the U5S form.
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Map of the Ints11 and Ints9 subunits of the integrator complex. The β‐lactamase and βCASP domains as well as the interaction domain between Ints11 and Ints9 are represented schematically. The vertical green bars represent conserved residues between Ints11 and CPSF73 and between Ints9 and CPSF100. The sequences of the motifs characteristic of the β‐lactamase and βCASP family (1–4 and A–C) are given for each protein. The amino acids responsible for the coordination of the zinc ions in the catalytic center of the proteins are represented in red. Note that most are absent in Ints9/CPSF100.
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Schematic of Saccharomyces cerevisiae U1 and U2 snRNA 3′ end formation pathways. (a) U1 snRNA utilizes both Rnt1‐dependent and Rnt‐independent mechanisms to achieve 3′ end formation. The RNAseIII, Rnt1, cleaves at a stem loop region generating a substrate for 3′ to 5′ exonucleases giving rise to the mature product, which can also be generated through termination followed by exonucleolytic trimming. A polyadenylated intermediate may also be formed that may cycle back to an exonuclease substrate. (b) U2 snRNA uses both Rnt1‐dependent and Rnt1‐independent mechanisms to generate its 3′ end. The Rnt1‐dependent is similar to the U1 snRNA 3′ end pathway but the Rnt1‐independent utilizes polyadenylation sequences (PASs) located downstream of the Rnt1 cleavage site. This generates a cleavage and polyadenylation substrate and ultimately poly(A)+ U2 snRNA.
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Model for metazoan U snRNA 3′ end processing. (a) Organization of a typical U snRNA gene. The distal element (DSE) and the proximal element (PSE) are located approximately 200 and 50 bp upstream of the start site respectively. The 3′ box is located 9–19 bp downstream of the mature end of the U snRNA. (b) Transcription initiation of U snRNA genes. The CTD of the polymerase is phosphorylated by CDK7 (ser5P and ser7P) and CDK9 (ser2P). RPAP2 binds specifically the ser7P mark. The Integrator Complex is in turn recruited to the carboxy‐terminal domain (CTD) through its interaction with RPAP2 and ser2P. The exact mechanism driving Integrator Complex specificity for U snRNA genes is unknown, as is the integrator subunit responsible for binding ser2P. C. U snRNA 3′ end recognition and cleavage. After transcription of the 3′ box, the Integrator Complex recognizes the sequence of the 3′ box in the nascent transcript and most probably the terminal stem‐loop of the U snRNA, triggering the cleavage by Ints11 of the pre‐U snRNA between these two elements. The identity of the proteins contacting the nascent U snRNA is currently unknown.
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RNA Processing > Processing of Small RNAs
RNA Processing > 3′ End Processing

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