Lecture 13 RNA Processing I

RNA processing I Side notes: Pre-RNAs can be modified in various ways RNAs are synthesized from DNA templates, but the molecules produced are often not immediately functional. These precursor RNAs (pre-RNAs) need to be modified to make the mature, functional RNA RNA processing has three main benefits Regulation - RNA processing offers another layer of gene regulation Diversity - many different RNAs can be produced from one gene via alternative splicing (by removal of different combinations of introns) Quality control - detective mRNAs are detected and degraded Top Hat 1 What is the advantage of processing multiple rRNAs from a single precursor RNA? • It maintains a balance between the copy numbers of ribosome components • It allows rRNAs to be exported to the cytoplasm individually and assembled into ribosomes independently • The precursor is resistant to enzymatic degradation compared to individual rRNA molecules RNA processing complexes often contain RNA components Many RNA processing complexes contain both protein and RNA (ribonucleoproteins; RNPs) The RNA in RNPs can be structural, but can also have catalytic activities (ribozymes) Some RNPs contain guide RNAs that base pair with pre-RNAs and guide the RNP to the correct place for processing tRNAs and rRNAs are made from longer precursors An E. coli precursor encodes three rRNAs and several tRNAs The S. cerevisiae precursor encodes three tRNAs Transcription and processing of eukaryotic rRNAs occur in the nucleolus Ribonucleases cleave and trim rRNA precursors Encoding several RNAs in one precursor ensures that similar amounts of each RNA are made tRNA and rRNA processing can involve many steps and multiple pathways The reasons why multiple pathways exist are not known Some rRNA introns can catalyze their own removal - they are self-splicing. The precursor RNAs therefore act as ribozymes themselves Ribonucleases cleave the RNAs into smaller parts Exonucleases successively remove nucleotides from the end of a transcript, most often in the 3' to 5' direction but sometimes 5' to 3' Exonucleases are not usually sequence specific Endonucleases cleave the DNA within the strand. Some are specific doublestranded RNA, some for single-stranded RNase III and RNase P are examples of endonucleases Ribonuclease P (RNase P) trims tRNA 5’ ends RNase P is an example of a ribozyme - it has an RNA component as well as proteins The bacterial RNA component alone can cut RNA, and the protein part enhances the activity and may determine substrate range The eukaryotic, archaeal and mitochondrial Rnase P RNA component cannot cut RNA alone, but is essential for function CCA is added to the ends of tRNAs The 3' ends of tRNAs have a conserved CCA sequence - this is the attachment site for the amino acid The CCA is sometimes encoded by the tRNA gene but is most often added later by a CCA - adding enzyme (CCA tRNA-nucleotidyltransferase) RNA Processing I Side notes: Addition of CCA is catalyzed by the nucleotide binding pocket The pocket has three different conformations, and these control whether a C or an A is added tRNA and rRNA nucleotides are often modified after transcription More than 80 modifications have been observed in tRNAs, and they increase the repertoire of shapes, structures, and stability of tRNA molecules Modifications can be small, like methylation, or larger, like addition of threonine The most common rRNA modifications are ribose 2'-O-methylation and pseudouridylation Many rRNA modifications are found in regions important for ribosome function and they are usually essential for growth and survival snoRNAs guide methylation and pseudouridylation The enzymes that catalyze ribose methylation and the uridine to pseudouridine transition are similar in an organisms, but the recognition mechanisms are different Eukaryotes and archaea use small nuclear RNAs (snoRNAs) to guide enzymes to the correct site. snoRNAs associate with a complex of proteins to make an RNP called snoRNP snoRNAs are found in the nucleolus, and are 60-300 nucleotides long. Most vertebrate snoRNAs are made from the introns of precursor mRNAs The snoRNA base-pairs with specific regions of target RNAs and directs enzymes to those positions snoRNAs that direct ribose methylation are box C/D snoRNAs. snoRNAs that direct pseudouridylation are H/ACA snoRNAs Eukaryotic mRNAs are capped at the 5' end End modifications protect eukaryotic mRNAs from nuclease degradation and help with protein interactions The 5' ends are capped with a 7-methylguanine nucleotide via a 5’-5’ triphosphate linkage. This guanine is then methylated at N7 The 5' cap is needed for efficient elongation and termination of the transcript, for mRNA processing and export from the nucleus, and for directing translation In more complex eukaryotes, the 2' O of the second and sometimes third base are methylated The 5' cap is added in three stages, shortly after the mRNA emerges from RNA pol II (about 20-30 nucleotides) First, an RNA 5' triphosphate catalyzes removal of a phosphate from the 5' end Second, a guanyl transferase attaches a guanosine monophosphate (GMP) to the end in a 5'- 5' triphosphate linkage Third, the guanine is methylated by a guanine-7 - methyl transferase Eukaryotic mRNAs are polyadenylated at the 3' end Polyadenylation at the 3' end of eukaryotic mRNAs starts with an initial cleavage This cleavage usually occurs after a CA that lies between a conserved AAUAAA hexameter and a U or GU - rich region After cleavage, ~200 adenosines are added by poly(A) polymerase RNA Processing I Side notes: Top Hat 2 5’ capping and poly (A) machinery are recruited by the RNA pol II CYD. Why does this ensure that only mRNAs are capped/ polyadenylated? • RNA pol I and RNA pol III are located in the cytoplasm, where capping and polyadenylation do not occur • RNA pol I and RNA pol III do not contain the same type of CTDs • RNA pol I and RNA pol III exclusively synthesize ribosomal RNA, which naturally contains a 5’ cap and poly (A) tail A larger protein complex is required for polyadenylation than for 5' capping, probably because it is more complex to recognize the different polyadenylation sites in different mRNAs The 3' end of most eukaryotic mRNAs have about 200 adenosines added this is a polyadenosine, or poly (A) tail mRNAs have polyadenylation sites, where pre-mRNAs are cleaved and the poly (A) tail added Multiple polyadenylation sites are found in some mRNAs, such as cyclin D1 mRNA above, and these can participate in regulation Polyadenylation at the distal site retains multiple regulatory sequences Polyadenylation at the proximal site eliminates the regulatory sequences 5’ capping and 3’ polyadenylation are linked to transcription through RNA pol II Capping is needed to allow RNA pol II to continue transcription. Polyadenylation is needed for efficient transcription termination The C-terminal domain (CTD) of the largest subunit of RNA polymerase II, RPB1, is responsible for mediating mRNA processing RNA pol II CTD sequentially recruits the different processing complexes: CTD becomes partially phosphorylated on transcription initiation and recruits capping enzyme Elongation leads to more phosphorylation of CTD, which recruits splicing machinery This also leads to recruitment of the change and polyadenylation complex Introns must be removed from pre-RNAs Most introns do not contain genes and are excised and degraded (exceptions are snoRNAs and certain miRNAs) Some introns are removed by proteins, some by RNPs, and some excise themselves. All mechanisms use transesterification reactions Introns are far more prevalent in eukaryotes than in bacteria Introns allow for exon shuffling, where exons are exchanged and reordered via recombination, allowing evolution of different genes. Differential removal of introns also gives different transcripts from the same gene Splicing occurs via sequential transesterification Most introns are removed by a complex called the spliceosome, but some are self-splicing Both steps involve transesterification reactions - a single phosphodiester bond is broken, and replaced by another phosphodiester bond of similar energy Group 1 self-splicing introns Group 1 introns are found in bacteria, viruses, microeukaryotes and plants The are ~ 120 - 450 nucleotides long Many (but not all) group 1 introns can excise themselves from the primary transcript In the first transesterification, a free guanosine is the attacking species that detaches the 5' end of the intron from exon 1 In the second reaction, the released end of exon 1 attacks the intron-exon 2 junction, joining the two exons together Splice sites are defined by the 3D structure of the intron and by recognition of a conserved G-U wobble pair RNA Processing I Side notes: Group II self-splicing introns Group II introns (~400-1000 nucleotides long) are in bacteria and in organelles genes of plants and fungi The 2' OH of a specific A within the intron attacks the exon 1 - intron junction Once released, the intron forms a branched lariat intermediate Group I introns can also act as mobile genetic elements by reverse splicing, and the often have open reading frames within the introns that assist splicing Splicing of eukaryotic mRNAs is performed by the spliceosome Eukaryote genes often have several introns, which can be very long (thousands of bases) and account for up to ~90% of a pre-mRNA The human dystrophin gene has 78 introns and exons are sometimes many thousands of bases apart Some eukaryotes, like yeast, have flower, shorter introns Most eukaryotic introns are not self-splicing, so splicing is mediated by the spliceosome, which is made of several small nuclear ribonucleoproteins (snRNP) Spliceosome catalyzed splicing is similar to that of group II introns Top hat 3 Why are introns much longer in the human genome than in the yeast genome? • Human genes have more complexity and more alternative splicing • Human introns contain more regulatory signals • Longer introns provide a layer of transcriptional delay • Human introns contain more repeats • Other The spliceosome recognizes splice sites in pre-mRNAs Splice sites are defined by short sequence motifs - the 5' and 3’ splice sites and a branch-point nucleotide within the intron (like the A in group II introns) and a polyprimidine region before the 3' splice site Usually, introns have GU and AG at their ends Other sequences help define intron-exon boundaries, and also help cells produce different mature mRNAs from the same gene Spliceosome assembly involves RNA-RNA interactions The spliceosome has 5 snRNPs (U1, U2, U4, U5, U6). Each has a snRNA of about 100 - 300 nucleotides, plus proteins The snRNAs form base-pairs with the pre- mRNA and work as the recognition part of the snRNP The 5' splice site is recognized by U1 and other sequences are bound by non-snRNPs The rest of the splicing apparatus binds, sometimes displacing other factors, including the branch - point- binding protein; BPP The pre-mRNA rearranges, the first transesterification occurs and the lariat forms Other rearrangements then bring the two exons together for the second transesterification The exon junction complex marks processed transcripts Some components of the human spliceosome are also involved in transcription, polyadenylation, nuclear mRNA and translation For example, the exon junction complex (EJC) is left at splice junctions after splicing The EJC marks the transcript as processed and interacts with export and translation proteins