Small RNAs and their regulatory roles. Presented by: Chirag Nepal

Papers to be discussed  Tiny RNAs associated with transcription start sites in animals. – Nat Genet May;41(5): Epub 2009 Apr 19.  Nuclear-localized tiny RNAs are associated with transcription initiation and splice sites in metazoans. – Nat Struct Mol Biol Aug;17(8): Epub 2010 Jul 11.

Introduction Small RNAs are involved in many biological and cellular processes. Previously discovered short RNAs that map to promoter region. – Yeast ( nt ) – Arabidopsis (variable size) – PASRs in mammalian transcriptome (<200 nt) Explore the nature of small RNAs that map to promoter regions. – Examined deep-sequencing libraries from human, chicken and Drosophila. – Identified a new class of small RNAs that are predominantly 18 nt in length, derived near the 5’ end of transcribed genes.

Identification of tiRNAs Are these tiRNAs degraded products ?

 RNAs are most abundant at -60 nt to +120 nt relative to TSS, mostly on the same strand as the TSS.  tiRNAs loci express bidirectional transcripts. Genomic Distribution of tiRNA  Identified 2,312 human tiRNAs.  Predominantly downstream of highly expressed deepCAGE tag in a promoter.  Identified 1,186 tiRNAs in chicken.  tiRNA discovery is dependent on the sequencing depth.

tiRNAs are not truncated or degraded 5’ ends of highly expressed capped transcripts. Found almost exclusively downstream of TSS.

Size distribution of tiRNAs tiRNAs at 5’ end tiRNAs at 3’ end

Gene and tiRNA expression  Relative gene expression of all human genes with tiRNA (1,318 tiRNAs, 947 genes) with 3,368 Refseq genes that do not have tiRNAs  Relative gene expression of chicken genes with tiRNA and without tiRNAs.  *P <0.01, **P <0.001, ***P<

tiRNA and deepCAGE abundance do not exhibit linear relationship

Gene Ontology terms are consistent with high expression.

Do promoters with tiRNAs show enrichment for genomic features of active transcription ?  Examine the loci for evidence of H3K9 acetylation, binding of RNA pol II, and TF Sp1 and Pu.1 in THP-1 cells.  Enrichment for binding of RNA pol II and Sp1.  No enrichment for H3K9 acetylation or PU.1 binding.

Discussion  A new class of small RNA, 18 nt in length, adjacent to TSS.  Show similarities with previously described PASRs  Low abundance, distribution skewed to 3’ of TSS  Occurrence in more than one organism  Associated with highly expressed genes and GC rich  But vary significantly in size length (70 nt).  Are tiRNAs simply signatures of transcription or have a particular function ?  Evolutionary conserved, non random distribution and distinct size characteristics indicate they are not random noise but a new and distinctive size of RNA centrally associated with transcription.

 High-throughput RNA sequencing have led to detection of new members of established classes of small RNAs and many novel RNAs.  Discovery and characterization of at least 3 classes of promoter proximal RNAs: – 5’ capped promoter-associated small RNAs (PASRs) – Transcription start site (TSS)-associated RNAs (Tssa RNAs) – Transcription initiation RNAs (tiRNAs)  Performed deep sequencing of small RNAs from the nuclear and cytoplasmic fractions of human monocytic leukemia cell (THP-1) and nuclei of primary mouse granulocytes. – Determine the sub cellular localization of tiRNAs and possibility of other classes of nuclear-enriched small RNAs.

tiRNAs are localized in the nucleus  tiRNAs are > 40 fold enriched in the nucleus.  nuclear RNAs (7,014 Refseq genes), cytoplasmic (914) Assessed relative nuclear enrichment of tiRNAs by analysis of THP-1 nucleus and cytoplasmic.

Genes with tiRNAs derived from both RNAs are more expressed.

Features of nuclear-localized small RNAs  Extensive S/AS cluster proximal to TSS  Peak tiRNAs and RNA Pol II density lies at the same position upstream of the +1 nucleosome.  tiRNAs show enrichment for 3’-terminal guanines in THP-1 cells.

Enrichment of 3’-terminal guanines even in chicken and Drosophila

tiRNAs are enriched at transcription-initiation chromatin marks

 Two fold enriched for chromatin marks (derived from CD4 + cells)

Splice-site RNAs are associated with splice donor sites  35 fold enriched in nuclear deep sequencing library (present in internal exons as well).  Splice-site RNAs are found in wide range of evolutionary distant metazoans.

MicroRNA-offset RNAs are nuclear enriched  MicroRNA-offset RNAs (moRNAs) are conserved small RNAs derived from the ends of pre-miRNAs.  moRNAs from 60 pre-miRNAs are mostly derived from 5’ arm, regardless of processed mature miRNAs, suggesting moRNAs and miRNA biogenesis may be linked but not interdependent.

Select miRNAs are nuclear enriched in THP-1 cells  Most miRNAs are cytoplasmically localized, while expression profiling revealed a small subset of miRNAs is also nuclear enriched.  Down regulation of the miR-15/16 cluster has been associated with chronic lymphocytic lymphoma, prostate carcinoma and targets multiple oncogenes, including BCL2, MCL1, CCND1 and WNT3A.  miR-15/16 might have additional nuclear functions or might interact with target within the nucleus.

sdRNAs show differential subcellular localization  Consistent with snoRNA localization to the nucleolus, C/D sdRNAs are 3- to 200-fold enriched in the THP-1 nuclear fraction.  However, sdRNAs from two H/ACA snoRNAs, SNORA36B and SNORA63, which are miRNA-like and are predominantly ~22 nt in length, are approximately 3-fold enriched in the cytoplasm.  These data indicate that the boundary between miRNAs and other small RNAs, particularly H/ACA sdRNAs, may be blurry.  snoRNAs, which are classified as either C/D or H/ACA, can be processed into snoRNA-derived RNAs (sdRNAs) with distinct size distributions.

Discussion  Wide range of small RNAs localized to, and abundant in the metazoan nucleus.  Many of these RNAs might be involved in regulating epigenomic modifications and transcription.

Thank you !